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Cytosolic sulfotransferases (SULTs) biotransform small molecules to polar sulfate esters as a means to alter their activities within the body. Understanding the molecular mechanism by which the SULTs perform their function is important for optimizing future therapeutic applications. Recent evidence suggests each SULT isoform acts by a half-site reaction (HSR) mechanism, in which a single SULT dimer subunit is active at any given time. HSR requires communication through the highly conserved KxxxTVxxxE dimerization motif. In this investigation, we sought to test the intersubunit interactions of SULT1B1 as it relates to enzyme activity. We generated two populations of SULT1B1 isoforms that efficiently heterodimerize upon mixing by targeted point mutation of the KxxxTVxxxE motif to KxxxTVxxxK or ExxxTVxxxE. The heterodimer exhibited wildtype-like activity with regard to native size, thermal integrity, PAP affinity, and PAPS Km, therefore serving as a valid model for investigating SULT1B1 dimer subunit interactions. The approach granted control over each independent subunit, permitting mutation of the critical 3′-phosphoadenosine 5′-phosphosulfate (PAPS) binding residue Arg258 and/or the catalytic base His109 in a single subunit of the dimer. Substitution of the dysfunctional subunits for fully active subunits yielded dimeric SULT1B1 with 50% the activity of the fully competent dimer, suggesting SULT1B1 intersubunit communication does not significantly contribute to the isoform’s activity. These results are a testament to the unique properties of individual SULT isoforms. The dimerization system described in this manuscript can be used to study subunit interactions in other SULT isoforms as well as proteins in other families.