Effects of HCO₃ on protein kinase C (PKC)- and protein kinase A (PKA)-induced anion conductances were investigated in Necturus gallbladder epithelial cells. In HCO₃-free media, activation of PKC via 12-O-tetradecanoylphorbol 13-acetate (TPA) depolarized apical membrane potential (V_a) and decreased fractional apical voltage ratio (F_R). These effects were blocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a Cl channel blocker. In HCO₃ media, TPA induced significantly greater changes in V_a and F_R. These effects were blocked only when NPPB was present in both mucosal and basolateral compartments. The data suggest that TPA activates NPPB-sensitive apical Cl conductance (g_Cl^a) in the absence of HCO₃; in its presence, TPA stimulated both NPPB-sensitive g_Cl^a and basolateral Cl conductance (g_Cl^b). Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased V_a and F_R; however, these changes were not affected by external HCO₃. We conclude that HCO₃ modulates the effects of PKC on g_Cl^b. In HCO₃ medium, TPA and IBMX also induced an initial transient hyperpolarization and increase in intracellular pH. Because these changes were independent of mucosal Na⁺ and Cl, it is suggested that TPA and IBMX induce a transient increase in apical HCO₃ conductance.