The present experiments demonstrated that the holotoxin as well as the A- and B-subunits of cholera toxin were able to directly enhance pregnenolone synthesis when isolated intact mitochondria, prepared from bovine adrenocortical tissue, were incubated; they were not, however, able to enhance pregnenolone synthesis when the inner mitochondrial fraction was similarly incubated, suggesting that the conformational structure of mitochondria is very important for activation of cholesterol side-chain cleavage by cholera toxin. Data are also presented demonstrating that cholera toxin can enhance Ca2+ release from isolated mitochondria, while pertussis toxin could activate neither pregnenolone generation nor increase Ca2+ efflux from mitochondria. Thus it is suggested that cholera toxin may activate pregnenolone synthesis by regulating Ca2+ movement in mitochondria.