Despite their broad application in the phosphoryl-transfer field, thio-nucleotides have not been available for the study of sulfate activation and transfer. There are two known forms of activated sulfate in the cell, APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). PAPS is the only known sulfuryl group donor in metabolism, and sulfuryl transfer is used widely to regulate metabolism. This study presents the first synthesis of a thio-nucleotide analogue of activated sulfate, APS(alpha)S ((Sp)-and (Rp)-adenosine 5'-O-(1-thiophosphosulfate)). Two syntheses are described, one of which is a novel "one-pot" method that is general for the site-specific delivery of the sulfuryl group. Both epimers of APS(alpha)S were purified and their stereochemical configurations were assigned. These compounds were used to address several stereomechanistic issues in the APS-synthesis reaction catalyzed by yeast ATP sulfurylase (ATP:sulfate adenylyl-transferase, EC 2.7.7.4). The reaction is shown to proceed with inversion of configuration at the alpha-phosphorus (P-alpha). The enzyme exhibits high selectivity for the (R-p) epimer of APS(alpha)S when Mg2+ is the activating cation. The "hard" vs "soft" cation dependence of the enzyme's activity indicates that its selectivity is due to cation coordination at P-alpha. The absence of selectivity in the substrate binding reactions indicates that coordination at P-alpha occurs after formation of the E.APS.PPi.M2+ complex.