The effect of aging was examined on cholinergically mediated contractions and acetylcholine (ACh) release in isolated colonic segments from Fischer (F344 x BN) F1 rats, 4-8 mo (postpubertal) and 22-28 mo (senescent) of age. This species demonstrates age-dependent slowing of colonic transit. Muscle tension response to electrical stimulation of cholinergic neural pathways and application of ACh was significantly decreased in preparations from senescent compared with postpubertal animals. We focused on the hypothesis that aging was associated with reduced ACh release that resulted from decreased calcium influx through membrane calcium channels. Aging did not affect either the synthesis of [3H]ACh from [3H]choline or the percentage of 3H released in the form of [3H]ACh. However, elevated KCl-evoked release of [3H]ACh was significantly reduced in tissue from senescent compared with postpubertal animals. Treatment with the calcium ionophore ionomycin increased [3H]ACh release in tissue from senescent animals to near postpubertal levels. However, increasing extracellular calcium concentration ([Ca2+]o) from 1.2 to 5 mM did not increase the amount of transmitter release in tissue from senescent animals to the levels observed with 1.2 mM [Ca2+]o in postpubertal tissue. The neuronal calcium channel antagonist omega-conotoxin GVIA inhibited acetylcholine release in a concentration-dependent manner with half-maximal inhibitory values of 1.8 and 8.2 nM for senescent and postpubertal preparations, respectively. In summary, age-dependent reduction in ACh release was observed in the rat colon myenteric plexus that may, in part, be associated with decreased calcium influx via membrane calcium channels.