Intracellular targets of the ubiquitous second messenger cAMP are located at great distances from the most widely studied source of cAMP, the G protein responsive transmembrane adenylyl cyclases. We previously identified an alternative source of cAMP in mammalian cells lacking transmembrane spanning domains, the "soluble" adenylyl cyclase (sAC). We now demonstrate that sAC is distributed in specific subcellular compartments: mitochondria, centrioles, mitotic spindles, mid-bodies, and nuclei, all of which contain cAMP targets. Distribution at these intracellular sites proves that adenylyl cyclases are in close proximity to all cAMP effectors, suggesting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases targeted to specific microdomains throughout the cell.