Membrane-bound mammalian adenylyl cyclase isoforms contain two internally homologous cytoplasmic domains (C1 and C2). When expressed separately, C1 and C2 are catalytically inactive, but conversion of ATP to cAMP is observed if C1 and C2 are combined. By analogy with DNA polymerases, adenylyl cyclases are thought to require two divalent metal ions for nucleotide binding and phosphodiester formation; however, only one Mg2+ ion (liganded to C1) has been visualized in the recently solved crystal structure of a C1-C2 complex [Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916]. Here, we have studied the binding of ATP to IIC2 (from type II adenylyl cyclase) using ATP analogues [2',3'-dialdehyde ATP (oATP), a quasi-irreversible inhibitor that is covalently incorporated via reduction of a Schiff base, the photoaffinity ligand 8-azido-ATP (8N3-ATP), and trinitrophenyl-ATP (TNP-ATP), a fluorescent analogue] and fluorescein isothiocyanate (FITC). [alpha-32P]oATP and 8N-[alpha-32P]ATP are specifically incorporated into IIC2. Labeling of IIC2 by [alpha-32P]oATP and by FITC is greatly enhanced by Mn2+ and to a much lesser extent by Mg2+. Similarly, TNP-ATP binds to IIC2 as determined by fluorescence enhancement, and this binding is promoted by Mn2+. Thus, a second metal ion binding site (preferring Mn2+) is contained within the C2 domain, and this finding highlights the analogy in the reaction catalyzed by DNA polymerases and adenylyl cyclases.