This study reports on the in vitro and in vivo two-photon luminescence (TPL) imaging of single gold nanorods. Gold nanorods, when excited at 830 nm, produce strong TPL signals with a cos⁴ dependence on the incident polarization. The TPL excitation spectrum overlaps with the longitudinal plasmon band, indicating plasmon-enhanced two-photon absorption. The TPL signal from a single nanorod is 58 times brighter than that from a single rhodamine molecule. In vivo imaging of single nanorods flowing in mouse ear blood vessels demonstrates the potential of gold nanorods as TPL imaging agents. Gold nanorods exhibit strong photoluminescence due to their plasmon resonances. The quantum efficiency of single-photon luminescence from gold nanorods is enhanced under plasmon-resonant conditions. Plasmon-resonant TPL is attractive for nonlinear optical imaging with 3D spatial resolution. Gold nanorods are particularly suitable as TPL substrates because their longitudinal plasmon modes are resonant at near-infrared wavelengths, where water and biological molecules have minimal absorption. Additionally, nanorods have larger local field enhancement factors than nanoparticles due to reduced plasmon damping. A two-photon excitation laser-scanning microscope was used to study plasmon-resonant TPL from single gold nanorods. The emission spectrum of the TPL was characterized as a function of excitation wavelength and polarization. The TPL signal from a single nanorod is essentially depolarized but has a cos⁴ dependence on the excitation polarization and is nearly 60 times brighter than the two-photon fluorescence from a single rhodamine molecule. These features make gold nanorods a unique imaging agent for multiphoton microscopy. In vivo imaging of gold nanorods in mouse ear blood vessels was demonstrated. The nanorods' plasmon resonance in the near-infrared region makes them ideal probes for TPL imaging of tissue samples. The TPL signal is resolved in the axial direction due to its nonlinear dependence on the excitation intensity. The intrinsic 3D spatial resolution of TPL is useful for monitoring biological processes in real time. The TPL intensity from individual nanorods is approximately three times that of the autofluorescence from the blood and surrounding tissue. The rapid clearance time suggests that gold nanorods will provide excellent TPL contrast when functionalized for cell-specific labeling.This study reports on the in vitro and in vivo two-photon luminescence (TPL) imaging of single gold nanorods. Gold nanorods, when excited at 830 nm, produce strong TPL signals with a cos⁴ dependence on the incident polarization. The TPL excitation spectrum overlaps with the longitudinal plasmon band, indicating plasmon-enhanced two-photon absorption. The TPL signal from a single nanorod is 58 times brighter than that from a single rhodamine molecule. In vivo imaging of single nanorods flowing in mouse ear blood vessels demonstrates the potential of gold nanorods as TPL imaging agents. Gold nanorods exhibit strong photoluminescence due to their plasmon resonances. The quantum efficiency of single-photon luminescence from gold nanorods is enhanced under plasmon-resonant conditions. Plasmon-resonant TPL is attractive for nonlinear optical imaging with 3D spatial resolution. Gold nanorods are particularly suitable as TPL substrates because their longitudinal plasmon modes are resonant at near-infrared wavelengths, where water and biological molecules have minimal absorption. Additionally, nanorods have larger local field enhancement factors than nanoparticles due to reduced plasmon damping. A two-photon excitation laser-scanning microscope was used to study plasmon-resonant TPL from single gold nanorods. The emission spectrum of the TPL was characterized as a function of excitation wavelength and polarization. The TPL signal from a single nanorod is essentially depolarized but has a cos⁴ dependence on the excitation polarization and is nearly 60 times brighter than the two-photon fluorescence from a single rhodamine molecule. These features make gold nanorods a unique imaging agent for multiphoton microscopy. In vivo imaging of gold nanorods in mouse ear blood vessels was demonstrated. The nanorods' plasmon resonance in the near-infrared region makes them ideal probes for TPL imaging of tissue samples. The TPL signal is resolved in the axial direction due to its nonlinear dependence on the excitation intensity. The intrinsic 3D spatial resolution of TPL is useful for monitoring biological processes in real time. The TPL intensity from individual nanorods is approximately three times that of the autofluorescence from the blood and surrounding tissue. The rapid clearance time suggests that gold nanorods will provide excellent TPL contrast when functionalized for cell-specific labeling.