A subset of myenteric neurons in the intestine (AH neurons) generate prolonged (>5 s) post-spike afterhyperpolarizations (slow AHPs) that are insensitive to apamin and tetraethylammonium. Generation of slow AHPs depends critically on Ca(2+) entry and intracellular release of Ca(2+) from stores, which then leads to the activation of a K(+) conductance that underlies the slow AHP (g(sAHP)). Slow AHPs are inhibited by stimulation of the cAMP/protein kinase A (PKA) pathway, suggesting that phosphorylation of the K(+)-channels that mediate the g(sAHP) (K(sAHP)-channels) is responsible for suppression of slow AHPs and possibly for the repolarization phase of slow AHPs. In the present study, we investigated the possibility that the rising phase of the slow AHP is mediated by dephosphorylation of K(sAHP)-channels by calcineurin (CaN), a Ca(2+)-calmodulin-dependent protein phosphatase, leading to an increase in g(sAHP) and activation of the associated current I(sAHP). Slow AHPs and I(sAHP) were recorded using conventional recording techniques, and we tested the actions of two inhibitors of CaN, FK506 and cyclosporin A, and also the effect of the CaN autoinhibitory peptide applied intracellularly, on these events. We report here that all three treatments inhibited the slow AHP and I(sAHP) (>70%) without significantly affecting the ability of neurons to fire action potentials. In addition, the slow AHP and I(sAHP) were suppressed by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results indicate that activation of the g(sAHP) that underlies the post-depolarization slow AHPs in AH neurons is mediated by the actions CaN and non-Ca(2+)-dependent phosphatases.