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 [Verkman et al., Am. J. Physiol. 250 (Cell Physiol. 19): C103-C114, 1986]. 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 10(3). 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 (graph see text) where H is hormone, R is receptor, C is catalytic unit, alpha and beta 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 alpha and beta subunits by providing a rapid pathway for addition of GTP to dissociated alpha subunits. The observed target size of the active enzyme species formed corresponds to the composite molecular weights of alpha GTP with C. This model consolidates the radiation inactivation findings as well as the known biochemical characteristics for adenylate cyclase.