PAPS (3′-phospho-adenosine-5′-phosphosulfate) synthases provide the cofactor PAPS for all human sulfation pathways. The cytoplasmic sulfotransferase SULT2A1 uses PAPS mainly to sulfate the androgen precursor DHEA (dehydroepiandrosterone). Apparent SULT2A1 deficiency is caused by mutations in the gene coding for PAPSS2; suggesting some form of PAPS synthase-sulfotransferase pairing. Knockdown studies within human adrenocortical NCI-295R cells now show that PAPSS2 is required for efficient DHEA sulfation, while PAPSS1 appears to be dispensable. As recombinant proteins, both PAPS synthases have similar specific activities in their APS kinase domains that catalyze the rate-limiting step of overall PAPS biosynthesis. DHEA sulfation rates in cells are significantly higher when cytoplasmic SULT2A1 is co-expressed with cytoplasmic PAPSS2, than any other localization variant. Proximity ligation assays between SULT2A1 and PAPSS2, and to a lesser extend also with PAPSS1, indicate a protein-protein interaction. Computational docking of PAPS synthases revealed a binding site for SULT2A1 within the APS kinase domain of PAPSS2. Energy-dependent scoring of various docking solutions identified the PAPSS2-SULT2A1 complex as more stable than the corresponding PAPSS1 complex; this interaction was also specific compared to the closely related SULT2B1 protein. This extended functional module within a human sulfation pathway may provide a better understanding of clinically observed PAPSS2 mutations.