Intracellular recordings were obtained from myenteric AH neurons of guinea pig ileum in vitro. Slow excitatory synaptic responses associated with decreased potassium conductance (gK), inhibition of the spike afterhyperpolarization current (AHC), and increased chloride conductance (gCl) were mimicked by senktide, a neurokinin3 receptor agonist. Intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) decreased gK and increased gCl irreversibly after nerve stimulation or senktide application. Myenteric neurons in pertussis toxin (PTX)-treated tissues responded normally to senktide and nerve stimulation. Forskolin and phorbol 12,13-dibutyrate (PDBu) inhibited gK and the AHC but did not activate gCl. The AHC was not reduced by subthreshold concentrations of forskolin (10 nM) or PDBu (3 nM) alone but was inhibited by forskolin and PDBu applied together. Inhibitors of phospholipase C (D-609) or protein kinases (staurosporine) reduced slow synaptic and senktide responses. The protein phosphatase inhibitor, calyculin A, caused an inward current, a decrease in gK, and AHC inhibition but did not activate gCl. We conclude that slow excitatory synaptic responses are mediated by PTX-insensitive G proteins and activation of phospholipase C and protein kinases. Forskolin and PDBu activate pathways that inhibit gK. The mechanisms for activation of gCl are unknown.