Mammalian ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase consists of kinase and sulfurylase domains, and catalyzes two sequential reactions to synthesize the universal sulfate donor, phosphoadenosine phosphosulfate (PAPS). In simpler organisms, the ATP sulfurylase and APS kinase reactions are catalyzed by separate enzymes encoded by two or three genes, suggesting that a fusion of separate genes during the course of evolution generated the bifunctional enzyme. We have characterized the genomic structure of the PAPS synthetase SK2 isoform genes for mouse (MSK2) and human (HSK2) and analyzed the possible fusion region. The MSK2 and HSK2 genes exhibit a common structure of 13 exons, including a 15-nucleotide alternatively spliced exon 8. Enzyme activities of several bacterially expressed exon assemblages showed exons 1-6 encode APS kinase, while exons 6-13 encode ATP sulfurylase. The MSK2 construct without the exon 6-encoded peptide showed no kinase or sulfurylase activity, demonstrating that exon 6 encodes sequences required for both activities. Exon 1 and its 5'-flanking sequence are highly divergent between the two species, and intron 1 of the HSK2 gene contains a region similar to the MSK2 promoter sequence, suggesting that it may be the remnant of a now-superceded regulatory region. The HSK2 promoter contains a GC-rich region, not present in the mouse promoter, and has few transcription factor binding sites in common with MSK2. These differences in the two promoter regions suggest that species-specific mechanisms regulate expression of the SK2 isoform.