Radiation inactivation is used to probe the sequence of subunit interactions involved in the activation of adenylate cyclase by vasopressin in cultured renal epithelial cells (LLC-PK1) based on our previous analysis of the radiation inactivation of multimeric enzymes. For basal adenylate cyclase activity, a concave downward ln(activity) vs. dose relation was observed with limiting slope corresponding to a molecular weight of (169-196) x 103. Similar results were obtained with NaF. In contrast, addition of vasopressin, guanylyl imidodiphosphate, or forskolin resulted in transition to a linear ln(activity) vs. dose relation with a slope corresponding to a molecular weight similar to that observed for basal activity. These findings were incorporated into a cyclic dissociation model for the hormonal activation of adenylate cyclase where H is hormone, R is receptor, C is catalytic unit, α and β are subunits of guanyl nucleotide-regulatory protein (G), GTP is guanosine triphosphate, and GDP is guanosine diphosphate. The addition of H favors the dissociation of G into α and β subunits by providing a rapid pathway for addition of GTP to dissociated α subunits. The observed target size of the active enzyme of α(GTP) with C. This model consolidates the radiation inactivation findings as well as the known biochemical characteristics for adenylate cyclase.