An approach for high-spatial-resolution mapping of functional circuitry in living mammalian brain slices has been developed. The locations of neurons making functional synaptic connections to a single neuron are revealed by photostimulation of highly restricted areas of the slice (50-100 microns in diameter) while maintaining a whole-cell recording of the neuron of interest. Photostimulation is achieved by bathing brain slices in a molecularly caged form of the neurotransmitter glutamate [L-glutamic acid alpha-(4,5-dimethoxy-2-nitrobenzyl) ester], which is then converted to the active form by brief pulses (< 1 ms in duration) of ultraviolet irradiation. Direct activation of receptors on recorded neurons in rat hippocampus and ferret visual cortex demonstrates that photostimulation is reliable and reproducible and can be repeated at the same site at least 30 times without obvious decrement in neuronal responsiveness. Photostimulation of presynaptic neurons at sites distant to the recorded neuron evoked synaptic responses in hippocampal and cortical cells at distances of up to several millimeters from the recorded neuron. Stimulation of 25-100 distinct presynaptic sites while recording from a single postsynaptic neuron was easily achieved. Caged glutamate-based photostimulation eliminates artifacts and limitations inherent in conventional stimulation methods, including stimulation of axons of passage, desensitization, and poor temporal resolution of "puffer" pipettes, and current artifacts of iontophoretic application. This approach allows detailed physiological investigation and manipulation of the complex intrinsic circuitry of the mammalian brain.