We identified the human ortholog of sAC (hsAC) in a locus linked to familial absorptive hypercalciuria and cloned it from a human cDNA library. hsAC transcripts are expressed in multiple tissues by RT-PCR and RNA blot. On RNA blot, a predominant 5.1kb band was detected in a multiple human tissue blot but three splice transcript variants were detected by RT-PCR and confirmed by sequence analysis. Immunoblot showed 190 kDa and 80 kDa bands in multiple human cell lines from gut, renal, and bone origins in both cytosol and membrane fractions: Caco2 colorectal adenocarcinoma, HEK293, HOS osteosarcoma, primary human osteoblast, and in vitro-induced osteoclasts. Specificity of the antiserum was verified by peptide blocking and reduction by sequence-specific siRNA. Confocal immunofluorescent cytochemistry localized hsAC primarily in cytoplasm but some labeling was observed in the nucleus and plasma membrane. Cytoplasmic hsAC co-localized with microtubules but not with microfilaments. To test the function of hsAC, 4 constructs containing catalytic domain I & II (aa1-802), catalytic domain II (aa231-802), non-catalytic domain (aa648-1610), and full length protein (aa1-1610) were expressed in sf9 insect cells. Only catalytic I & II or full length proteins showed adenylyl cyclase activity. Mg2+, Mn2+, and Ca2+ all increased cyclase activity in a dose-dependent manner. While hsAC had minimal response to HCO3- in the absence of divalent cations, HCO3- robustly stimulated Mg2+-bound hsAC but inhibited Mn2+-bound hsAC in a dose-dependent manner. In summary, hsAC is a divalent cation and HCO3- sensor and its HCO3- sensitivity is modulated by divalent cation.