In monkey dorsolateral prefrontal cortex (PFC), long-distance, horizontally oriented intrinsic axon collaterals interconnect clusters of pyramidal neurons in the supragranular layers. In order to study the electrophysiological responses mediated by these long-distance projections, an in vitro slice preparation of monkey PFC was used to obtain whole-cell patch clamp recordings from layer 3 pyramidal neurons. Using in vivo tracer injections, we found that long-distance projections were well preserved in PFC slices cut in the coronal plane. Postsynaptic currents were evoked by low-intensity electrical extracellular stimulation applied successively to 20-30 discrete sites located up to 2200 micron lateral to the recorded cell. Several criteria were applied to discriminate between mono- and polysynaptic responses. Long-distance monosynaptic connections were mediated by fibers with relatively slow conduction velocity (0.14 m/s). Excitatory postsynaptic currents (EPSCs) evoked by stimulation of short- or long-distance horizontal connections did not differ in kinetic properties. The majority (77%) of the 35 layer 3 PFC neurons studied were monosynaptic targets of long-distance connections. EPSCs mediated by long-distance connections had amplitudes that were similar or even larger than short-distance EPSCs, suggesting that excitatory input provided by the former was relatively robust. For most neurons (87.5%) in which a full complement of monosynaptic EPSCs was evoked by multisite stimulation, the EPSC amplitude as a function of stimulation distance from the recorded cells exhibited statistically significant peaks. The spacing between peaks was similar to the spacing between interconnected clusters of neurons observed in previous anatomical studies. The results show that long-distance excitatory connections constitute a significant intrinsic pathway of synaptic communication in layer 3 of monkey PFC.