We investigated the regulation of Ca²⁺-activated Cl⁻ channels in cells from the human colonic cell line T84 and acinar cells from rat parotid glands. The participation of multifunctional Ca²⁺- and calmodulin-dependent protein kinase (CaM kinase) II in the activation of these channels was studied using selective inhibitors of calmodulin and CaM kinase II. Ca²⁺-dependent Cl⁻ currents were recorded using the whole cell patch-clamp technique. Direct inhibition of CaM kinase II by 40 μM peptide 281–302 or by 10 μM KN-62, another CaM kinase inhibitor, did not block the Cl⁻ current in parotid acinar cells, whereas in T84 cells KN-62 markedly inhibited the Ca²⁺-dependent Cl⁻ current. We also used the calmodulin-binding domain peptide 290–309 (0.5 μM), which competitively inhibits the activation of CaM kinase II. This peptide reduced the Cl⁻ current in T84 cells by ∼70% but was without effect on the channels in parotid acinar cells. We conclude that the Ca²⁺-dependent Cl⁻ channels in T84 cells are activated by CaM kinase II but that the channels in parotid acinar cells must be regulated by a fundamentally different Ca²⁺-dependent mechanism that does not utilize CaM kinase II or any calmodulin-dependent process.