Ganglion cells of the myenteric plexus of the guinea pig were physiologically classified as to cell type using intracellular microelectrodes containing horseradish peroxidase (HRP). Interganglionic fiber tracts were then stimulated in an attempt to elicit slow excitatory postsynaptic potentials (EPSPs) in the impaled cells. The presence or absence of a slow EPSP was noted, following which the cells were injected with HRP through the recording micropipette and finally were incubated with tritiated 5- hydroxytryptamine ([3H]-5-HT; 0.5 microM) and desmethylimipramine (10 nM). The preparations were examined by light and electron microscopy (EM) for which the physiologically identified, HRP-injected neurons were demonstrated histochemically and serotonergic nerve terminals were simultaneously demonstrated radioautographically. Forty-seven cells were physiologically identified, injected with HRP, and studied by light microscopy. Of these, 22 were also fully analyzed by electron microscopy. The sample included 13 type I/S cells (6 analyzed by electron microscopic radioautography), 16 type II/AH cells (10 analyzed by electron microscopic radioautography), and 18 nonspiking (NS) cells (6 analyzed by electron microscopic radioautography). Slow EPSPs were only observed in type II/AH cells. All five of the fully analyzed subset of type II/AH cells that manifested a slow EPSP were contacted by serotonergic terminals. These terminals were found on 7 of 10 type II/AH cells, 2 of 6 type I/S cells, and 0 of 6 NS cells. Serotonergic terminals, therefore, contacted type II/AH cells (p less than 0.05) and especially those that showed a slow EPSP (p less than 0.005) more frequently than other types of ganglion cells. Morphologically, cells with short, stubby dendrites were reproducibly found to be type I/S cells, and glia could be recognized by their astrocytic appearance; however, cell shape did not otherwise correlate with physiological cell type. These data are compatible with and provide support for the hypothesis that 5-HT is one of the mediators of slow EPSPs in the myenteric plexus.