cAMP is a ubiquitous second messenger produced from ATP and involved in many cellular processes. In humans, cAMP is produced from two types of adenylyl cyclases: G protein-regulated transmembrane adenylyl cyclases (ADCY1-9) and bicarbonate- (HCO3-) and calcium- (Ca2+) regulated soluble adenylyl cyclase (sAC; ADCY10). sAC is molecularly and biochemically distinct from other mammalian nucleotidyl cyclases. In most mammals, sAC arises from a single gene which is predicted to generate multiple isoforms via alternative splicing. In rodents, there are two molecularly identified splice variants: the "full-length" isoform (sACfl) and a "truncated" isoform (sACt). To date, biochemical and structural characterization of sAC has focused almost exclusively on the sACt isoform. Longer sAC isoforms, including the longest known sACfl, contain additional presumptive regulatory domains which have not yet been functionally characterized. Thus far, studies have been limited by the inability to obtain sufficient sACfl protein to allow in vitro biochemical characterization. Here, we describe attempts to heterologously express and purify human sACfl as well as generation of a novel genetically modified mouse strain which permits biochemical separation and purification of endogenously expressed mouse sACfl and sACt. We use these heterologously expressed and endogenous proteins to compare and contrast the biochemically properties of human and mouse sACfl and sACt.