It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl conductance (G_Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G_Cl is independent of protein kinase A. We activated G proteins and monitored CFTR G_Cl in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTR G_Cl in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR G_Cl required Mg²⁺ and ATP hydrolysis (5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR G_Cl was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 µM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 µM); and 3) independent of Ca²⁺, suggesting that Ca²⁺-dependent protein kinase C and Ca²⁺/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10⁶ M forskolin plus 10⁶ M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR G_Cl endogenously via a cAMP-independent pathway in this native absorptive epithelium.