Regulation of multiple adenylyl cyclases (AC) provides unique inputs to mediate the synthesis of cAMP, a ubiquitous second messenger that controls many aspects of cellular function. On stimulation by G(s), the activities of ACs can be further selectively modulated by other pathways to ensure precise control of intracellular cAMP responses to specific stimuli. Recently, we reported that one of the AC isoforms, AC7, is uniquely regulated by the G(13) pathway. To understand more fully the molecular mechanism of this regulation, we compared the regulation of AC7 with that of AC2 in bone marrow-derived macrophages devoid of AC7. Although both enzymes could fully restore regulation of cAMP by Gβγ, activation of the G(13) pathway preferentially synergized with AC7. Exchange of domains between the two isoforms indicates that the C1b domain and the N-terminus of the C1a domain are important for directing selective regulation of AC7 by the G(13) pathway. A mutagenesis screen identified more specific regions of AC7 that differentially mediate its regulation by distinct pathways.