Melittin, a surface-active, 26-amino acid polypeptide from bee venom, has been reported to alter a variety of membrane properties including stability, permeability, and fluidity, the latter having been shown to be altered in a biphasic manner. Melittin induced a biphasic alteration of rat heart microsomal adenylate cyclase activity, stimulating it at low concentration (<30 μg/ml) and inhibiting it at higher concentrations (100 μg/ml or higher). Melittin potentiated sodium fluoride and 5′-guanylylimidodiphosphate activation of adenylate cyclase below 40 μg/ml but it inhibited at high concentrations, except in the presence of high concentrations of 5′-guanylylimidodiphosphate (10−4m). Basal and fluoride-activated adenylate cyclase exhibited no significant change in the Km for ATP in the presence of melittin at <40 μg/ml, but the V was elevated. Potentiation by melittin of adenylate cyclase was observed at all fluoride, 5′-guanylylimidodiphosphate, and magnesium concentrations tested. The observed effects of melittin on rat heart adenylate cyclase are consistent with it acting by altering the properties of membrane lipids with which the enzyme is associated.