A novel in vivo method was developed to measure the activity of direct- and indirect pathways in the striatum of mice. Using Cre-dependent viral expression of the genetically encoded calcium indicator GCAMP3 in D1-Cre and A2A-Cre mice, the study observed transient increases in neural activity in both pathways during action initiation, but not during inactivity. These findings challenge the classical model of basal ganglia function, suggesting that both pathways are co-activated during movement initiation, rather than opposing each other. The study also showed that neuronal activation in both pathways preceded movement initiation and predicted the occurrence of specific movements within 500 ms. These results indicate that coordinated activation of both pathways is important for action selection and precise timing of basal ganglia output. The study used time-correlated single photon counting (TCSPC) fiber optics to monitor fluorescence changes in GCAMP3, which reflects intracellular calcium transients in specific subgroups of neurons. The findings suggest that the predominant model of basal ganglia function, which posits more activity in the direct pathway during movement and more activity in the indirect pathway during immobility, may be incorrect. The study also highlights the importance of monitoring both pathways in behaving animals to understand the origin of motor symptoms in basal ganglia disorders. The results have implications for understanding the role of the basal ganglia in motor control and may inform future research on the treatment of movement disorders.A novel in vivo method was developed to measure the activity of direct- and indirect pathways in the striatum of mice. Using Cre-dependent viral expression of the genetically encoded calcium indicator GCAMP3 in D1-Cre and A2A-Cre mice, the study observed transient increases in neural activity in both pathways during action initiation, but not during inactivity. These findings challenge the classical model of basal ganglia function, suggesting that both pathways are co-activated during movement initiation, rather than opposing each other. The study also showed that neuronal activation in both pathways preceded movement initiation and predicted the occurrence of specific movements within 500 ms. These results indicate that coordinated activation of both pathways is important for action selection and precise timing of basal ganglia output. The study used time-correlated single photon counting (TCSPC) fiber optics to monitor fluorescence changes in GCAMP3, which reflects intracellular calcium transients in specific subgroups of neurons. The findings suggest that the predominant model of basal ganglia function, which posits more activity in the direct pathway during movement and more activity in the indirect pathway during immobility, may be incorrect. The study also highlights the importance of monitoring both pathways in behaving animals to understand the origin of motor symptoms in basal ganglia disorders. The results have implications for understanding the role of the basal ganglia in motor control and may inform future research on the treatment of movement disorders.