Peptide A1 of Vibrio cholerae toxin, nicotinamide adenine dinucleotide, adenosine triphosphate, and a soluble protein present in erythrocyte supernatant are required for the activation of pigeon erythrocyte ghost adenylate cyclase but are not required to maintain the activated state. The compounds are all required simultaneously, and when all are added to ghosts, adenylate cyclase activity increases at a linear rate without delay. Under optimal conditions significant activation of cyclase is given by less than one molecule of toxin per ghost. Intact cholera toxin may be inactive in vitro. There is a delay of about 1 min between the addition of intact toxin and the attainment of the final rate of increase of adenylate cyclase activity. During this period, glutathione reduces the disulfide bond between peptides A1 and A2. The delay is eliminated if the toxin is reduced before addition. More A1 is liberated if the toxin is also denatured with sodium dodecyl sulfate.