Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal motility. These peptides cause a variety of postjunctional responses including membrane hyperpolarization and inhibition of contraction. Neuropeptides released from enteric motor neurons can elicit responses by direct stimulation of smooth muscle cells as opposed to other transmitters that rely on synapses between motor nerve terminals and interstitial cells of Cajal. Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP(1–38) with confocal microscopy and patch-clamp technique. Localized Ca²⁺ transients (Ca²⁺ puffs) were observed in colonic myocytes, and these events coupled to spontaneous transient outward currents (STOCs). VIP and PACAP increased Ca²⁺ transients and STOC frequency and amplitude. Application of dibutyryl cAMP had similar effects. The adenylyl cyclase blocker MDL-12,330A alone did not affect spontaneous Ca²⁺ puffs and STOCs but prevented responses to VIP. Disruption of A-kinase-anchoring protein (AKAP) associations by application of AKAP St-Ht31 inhibitory peptide had effects similar to those of MDL-12,330A. Inhibition of ryanodine receptor channels did not block spontaneous Ca²⁺ puffs and STOCs but prevented the effects of dibutyryl cAMP. These findings suggest that regulation of Ca²⁺ transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of Ca²⁺ transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels.