J. Neurochem. (2010) 114, 1344–1352. Abstract Optogenetic control over neuronal firing has become an increasingly elegant method to dissect the microcircuitry of mammalian brains. To date, examination of these manipulations on neurotransmitter release has been minimal. Here we present the first in‐depth analysis of optogenetic stimulation on dopamine neurotransmission in the dorsal striatum of urethane‐anesthetized rats. By combining the tight spatial and temporal resolution of both optogenetics and fast‐scan cyclic voltammetry we have determined the parameters necessary to control phasic dopamine release in the dorsal striatum of rats in vivo. The kinetics of optically induced dopamine release mirror established models of electrically evoked release, indicating that potential artifacts of electrical stimulation on ion channels and the dopamine transporter are negligible. Furthermore a lack of change in extracellular pH indicates that optical stimulation does not alter blood flow. Optical control over dopamine release is highly reproducible and flexible. We are able to repeatedly evoke concentrations of dopamine release as small as a single dopamine transient (50 nM). An inverted U‐shaped frequency response curve exists with maximal stimulation inducing dopamine effluxes exceeding 500 nM. Taken together, these results have obvious implications for understanding the neurobiological basis of dopaminergic‐based disorders and provide the framework to effectively manipulate dopamine patterns.