Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal (GI) motility. These peptides cause a variety of post-junctional 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 apposed to other transmitters that rely upon synapses between motor nerve terminals and interstitial cells of Cajal (ICC). Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP (1-38) using confocal microscopy and the 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 similar effects as MDL 12,330A. Inhibition of ryanodine receptor channels did not block spontaneous calcium puffs and STOCs, but prevented the effects of dibutyryl-cAMP. These findings suggest that regulation of calcium transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of calcium transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels.