1. Pressure sensitive (P) neurons of the leech Hirudo medicinalis show both an inhibitory, Cl(-)-dependent response and a depolarizing, cationic response to pipette application of serotonin (5-HT). Serotonergic Retzius (R) neurons in culture reform inhibitory, Cl(-)-dependent synapses with P neurons but fail to elicit the extrasynaptic, depolarizing response to 5-HT. We have examined the localization of the selection of 5-HT responses by testing the sensitivity of P cell growth cones and neurites to 5-HT application. 2. As measured by intracellular recording at the P cell soma, synaptic release of 5-HT from R cell processes activated only the Cl(-)-dependent response in P cell neurites. Focal application of 5-HT from a micropipette depolarized uncontacted P cell growth cones and neurites. In contrast, processes from the same P cells that were contacted by R cells were rarely depolarized by 5-HT application unless the application pipette was moved along the neurites away from the sites of contact. 3. The channels underlying the depolarizing response to 5-HT were identified in patch clamp recordings from P cell growth cones. These cation channels showed rare, brief openings in the absence of 5-HT. Application of 5-HT in the bath (outside the patch pipette) increased channel activity in uncontacted P cell growth cones but not in growth cones of the same P cells contacted by R cells. 4. We conclude that the selection of transmitter responses during synapse formation was localized to discrete sites of contact between the synaptic partners.