In mammals, the second messenger cAMP is synthesized by a family of transmembrane isoforms (tmACs) and one known cytoplasmic enzyme, “soluble” adenylyl cyclase (sAC). Understanding the individual contributions of these families to cAMP signaling requires tools which can distinguish them. Here, we describe the structure-based development of isoform discriminating AC inhibitors. Docking calculations using a library of small molecules with the crystal structure of a sAC homologue complexed with the noncompetitive inhibitor catechol estrogen identified two novel inhibitors, 3,20-dioxopregn-4-en-21-yl 4-bromobenzenesulfonate (2) and 1,2,3,4,5,6,7,8,13,13,14,14-dodecachloro-1,4,4a,4b,5,8,8a,12b-octahydro-11-sulfo-1,4:5,8-dimethanotriphenylene-10-carboxylic acid (3). In vitro testing revealed that 3 defines a novel AC inhibitor scaffold with high affinity for human sAC and less inhibitory effect on mammalian tmACs. 2 also discriminates between sAC and tmACs, and it appears to simultaneously block the original binding pocket and a neighboring interaction site. Our results show that compounds exploiting the catechol estrogen binding site can produce potent, isoform discriminating AC inhibitors.