Target analysis of the turkey erythrocyteadenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system showed that the molecular weight of the ground state enzyme increases from 92,000 with MnATP as substrate and no stimulatory ligands to 226,000 when activated by fluoride ion or by 5'-guanyl imidodiphosphate (p[NH]ppG) subsequent to clearance of previously bound GDP. The identical increment in size (130,000) suggests that the same regulatory unit is.involved in the activation by-both effectors. When assayed with isoproterenol and p[NH]ppG, the enzyme system displayed a further increment in size of 90,000 daltons. Based on binding of the antagonist '25I-labeled hydroxybenzylpindolol,' the f-adrenergic receptor is about 90,000 daltons or the same as that seen for activation of the enzyme by isoproterenol through the j3-adrenergic-receptor. Because single targets were seen for the ground state enzyme system under all conditions, it would appear that the various regulatory and catalytic components are structurally linked prior to activation by hormone, guanine nucleotides, and fluoride ion. Furthermore, based on reported subunit sizes of the nucleotide regulatory and receptor components, it would appear from target analysis that these components are composed of multiple subunits, either homologous or heterologous in structure.A characteristic feature of hormone-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] systems is that they consist of at least three macromolecular components: hormone receptor (R), nucleotide regulatory component (N), and the catalytic unit (CU) that converts ATP to cyclic AMP (for recent review see ref. 1). Complexes formed between R and N and between N and CU have been obtained in detergent extracts ofmembranes (2-4). A fundamental question is: How are the three components organized in their normal membrane environment prior to and subsequent to activation of the enzyme system by hormones, guanine nucleotides, and fluoride ion?Target size analysis after irradiation with high-energy electrons has proved to be a useful technique for approaching this question (5, 6). In the case of the adenylate cyclase system, expression of activity and functional size can be affected differentially depending on the form of substrate and the presence of regulatory ligands that promote activation of the enzyme. For example, MnATP is the preferred substrate of CU in the absence of activating ligands and its conversion to cyclic AMP is thought to be a measure of the CU unit (7,8). The action of fluoride and 5'-guanyl imidodiphosphate (p[NH]ppG) is mediated by N-CU (8-11), whereas hormone action requires R in addition (12). As shown recently with the liver adenylate cyclase system (5), promotion of activity by fluoride ion, guanine nucleotides, or a combination of glucagon and guanine nucleotides leads to increasing sizes of the target, suggesting the involvement of the N and R units in the expression of catalytic activity. Higher-order structures were detected ...