The class III antiarrhythmic drugs amiodarone and bretylium tosylate are cationic/amphiphilic, and various substances with these physico-chemical properties are known to directly activate heterotrimeric regulatory G proteins. We asked the question of whether class III antiarrhythmic drugs are also direct G protein activators, using HL-60 leukemic cells and purified bovine brain G proteins as model systems. In HL-60 cell membranes, aminodarone increased high affinity GTP hydrolysis with an EC50 of 7.5 microM. The stimulatory effect of amiodarone on GTP hydrolysis was inhibited by pertussis toxin. Amiodarone stimulated binding of guanosine-5'-O-(3-thio)triphosphate to, and incorporation of GTP azidoanilide into, Gi protein alpha subunits in HL-60 membranes. The drug increased the cytosolic Ca2+ concentration in HL-60 cells in the presence but not in the absence of extracellular Ca2+. Amiodarone-induced increases in the cytosolic Ca2+ concentration were reduced by pertussis toxin and by a blocker of non-selective cation channels, SK&F 96365. Amiodarone activated the GTPase of reconstituted Gi/G(o) proteins and G12 with EC50 values of 20 microM and 50 microM, respectively. Bretylium tosylate did not increase GTP hydrolysis in HL-60 membranes or with Gi/G(o) proteins. Our data suggest that amiodarone but not bretylium tosylate is a direct activator of Gi and G(o) proteins and that amiodarone activates nonselective cation channels in HL-60 cells via Gi proteins and independently of Ca2+ mobilization from intracellular stores. Future studies will have to test the hypothesis that direct G protein activation by amiodarone contributes to its toxic and/or therapeutic effects.