The superior colliculus (SC) is a midbrain structure that plays a role in converting sensation into action. Most SC research focuses on either in vivo extracellular recordings from behaving monkeys or patch-clamp recordings from smaller mammals in vitro. However, the activity of neuronal circuits is necessary to generate behavior, and neither of these approaches measures the simultaneous activity of large populations of neurons that make up circuits. Here, we describe experiments in which we measured changes in membrane potential across the SC map using voltage imaging of the rat SC in vitro. Our results provide the first high temporal and spatial resolution images of activity within the SC. Electrical stimulation of the SC evoked a characteristic two-component optical response containing a short latency initial-spike and a longer latency after-depolarization. Single-pulse stimulation in the superficial SC evoked a pattern of intralaminar and interlaminar spread that was distinct from the spread evoked by the same stimulus applied to the intermediate SC. Intermediate layer stimulation produced a more extensive and more ventrally located activation of the superficial layers than did stimulation in the superficial SC. Together, these results indicate the recruitment of dissimilar subpopulations of circuitry depending on the layer stimulated. Field potential recordings, pharmacological manipulations, and timing analyses indicate that the patterns of activity were physiologically relevant and largely synaptically driven. Therefore, voltage imaging is a powerful technique for the study of spatiotemporal dynamics of electrical signaling across neuronal populations, providing insight into neural circuits that underlie behavior.