The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca²⁺ (Ca²⁺_i) have been investigated using inhibitors of CaM and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased following the rapid and sustained increase in Ca²⁺_i (to micromolar concentrations) observed following addition of ionophore plus Ca²⁺, but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed following the addition of ATP that we have reported previously ([8] Churchill GC et al, AJP 281: C972-C981, 2001.) could be prevented by either the direct or indirect inhibition of PKC, but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 following this ATP-dependent transient decrease in cell-to-cell coupling. While BAPTA-loaded cells did not display the ATP-dependent transient increase in Ca²⁺_i, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Ca²⁺_i-independent. Thus, CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Ca²⁺_i concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist-activation of second messenger pathways that are independent of changes in Ca²⁺_i.