The activity of a calcium-activated nonselective (Ca-NS+) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, beta-NAD+, beta-NADH, beta-NADP+ and beta-NADPH were very similar when tested at 0.1 mM, and at 1 mM the phosphorylated forms, beta-NADP+ and beta-NADPH, appeared to be slightly more potent than beta-NAD+ and beta-NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch. The application of beta-NAD+, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the beta-NAD(+)-NS+ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.