A new pair of channelrhodopsins, Chronos and Chrimson, have been developed to enable independent optical activation of distinct neural populations in mammalian brain tissue. Chronos is a blue- and green-light drivable channelrhodopsin with faster kinetics than any previously described, while Chrimson is a red-light drivable channelrhodopsin with a spectral peak 45 nm more red-shifted than any previous channelrhodopsin. Together, these two reagents enable crosstalk-free two-color activation of neural spiking and downstream synaptic transmission in independent neural populations in mouse brain slices. Chronos represents an excellent general-use channelrhodopsin, while Chrimson enables temporally precise experiments requiring red light, such as deep tissue targeting or scenarios where blue light is visually distracting. The paper reveals tools of fundamental importance for many new neuroscientific experimental realms, and also provides new channelrhodopsins that may serve as protein backbones for future tools. The study demonstrates that Chrimson can be used in Drosophila experiments without inducing visually driven behavioral artifacts, and that it can be used for in vivo scenarios desiring light penetration without visual drive. The study also shows that Chronos and Chrimson can be used to independently drive synaptic transmission in mouse brain slices, with Chronos being able to reliably drive synaptic events in response to blue light and Chrimson reliably driving synaptic events in response to red light. The study also shows that Chronos has a higher effective light sensitivity than ChR2 and can be used to reliably drive neural spiking across a range of expression levels without altering neural excitability. The study also shows that Chrimson can be used for in vivo experiments with light powers that fall within the defined windows, which may require alternative illumination methods such as 3D optical waveguides or wireless LED implants. The study also shows that the temporal precision of Chronos and Chrimson mediated synaptic events may depend on light power, potentially limiting usage in scenarios requiring sub-millisecond timing of synaptic release. The study also shows that the stimulation frequency for both red and blue pulses is fundamentally limited by the wild-type Chrimson, the slowest of the opsin pair. The study also shows that ChrimsonR, a faster Chrimson kinetic mutant, has similar blue light sensitivity to the wildtype but allows modulation at >20Hz range. The study also shows that the photosensitivity of the fly eye extends well beyond the wavelength of typical room light for fly experiments, highlighting the utility of the 720 nm stimulation protocol for a wider range of behavioral experiments. The study also shows that wavelengths lower than 720 nm may be useful in situations where startle responses do not affect measurements of the parameters under study. The study also shows that the use of Chronos and Chrimson allows for reliable Chronos-induced spikes with zero Chrimson-induced spikes in mouse corticalA new pair of channelrhodopsins, Chronos and Chrimson, have been developed to enable independent optical activation of distinct neural populations in mammalian brain tissue. Chronos is a blue- and green-light drivable channelrhodopsin with faster kinetics than any previously described, while Chrimson is a red-light drivable channelrhodopsin with a spectral peak 45 nm more red-shifted than any previous channelrhodopsin. Together, these two reagents enable crosstalk-free two-color activation of neural spiking and downstream synaptic transmission in independent neural populations in mouse brain slices. Chronos represents an excellent general-use channelrhodopsin, while Chrimson enables temporally precise experiments requiring red light, such as deep tissue targeting or scenarios where blue light is visually distracting. The paper reveals tools of fundamental importance for many new neuroscientific experimental realms, and also provides new channelrhodopsins that may serve as protein backbones for future tools. The study demonstrates that Chrimson can be used in Drosophila experiments without inducing visually driven behavioral artifacts, and that it can be used for in vivo scenarios desiring light penetration without visual drive. The study also shows that Chronos and Chrimson can be used to independently drive synaptic transmission in mouse brain slices, with Chronos being able to reliably drive synaptic events in response to blue light and Chrimson reliably driving synaptic events in response to red light. The study also shows that Chronos has a higher effective light sensitivity than ChR2 and can be used to reliably drive neural spiking across a range of expression levels without altering neural excitability. The study also shows that Chrimson can be used for in vivo experiments with light powers that fall within the defined windows, which may require alternative illumination methods such as 3D optical waveguides or wireless LED implants. The study also shows that the temporal precision of Chronos and Chrimson mediated synaptic events may depend on light power, potentially limiting usage in scenarios requiring sub-millisecond timing of synaptic release. The study also shows that the stimulation frequency for both red and blue pulses is fundamentally limited by the wild-type Chrimson, the slowest of the opsin pair. The study also shows that ChrimsonR, a faster Chrimson kinetic mutant, has similar blue light sensitivity to the wildtype but allows modulation at >20Hz range. The study also shows that the photosensitivity of the fly eye extends well beyond the wavelength of typical room light for fly experiments, highlighting the utility of the 720 nm stimulation protocol for a wider range of behavioral experiments. The study also shows that wavelengths lower than 720 nm may be useful in situations where startle responses do not affect measurements of the parameters under study. The study also shows that the use of Chronos and Chrimson allows for reliable Chronos-induced spikes with zero Chrimson-induced spikes in mouse cortical