The RB cells, an identified cluster of serotonergic neurons in the abdominal ganglion of Aplysia californica, are excited by ACh. We have found that serotonin (5-HT) enhances the ACh responses recorded from the RB cells. The locus and specificity of the serotonergic interaction with the ACh response were analyzed using the conventional two-electrode voltage-clamp technique. We compared the current responses evoked in RB cells by constant-current iontophoretic pulses of ACh in control solutions and in solutions containing various concentrations of 5-HT. Serotonin consistently enhances the ACh evoked response in a dose-dependent manner while simultaneously directly activating the RB cells by turning on a small steady-state inward current. The dose-response characteristics for the 5-HT-mediated steady-state current differ, however, from those of the 5-HT-induced ACh response facilitation. The ACh response enhancement appears to be an effect of 5-HT upon RB cell ACh receptors since the enhancement is present under conditions that eliminate presynaptic input, and 5-HT is still capable of enhancing the iontophoretically evoked response when the AChE-resistant agonist carbachol is substituted for ACh. We tested other neurons in the Aplysia CNS exhibiting ACh evoked responses qualitatively similar to the RB cell ACh responses; 5-HT does not enhance the iontophoretically evoked ACh responses of these cells. Also, although the RB cells are responsive to dopamine and histamine, these putative neurotransmitters do not enhance the ACh response.