ATP-sulfurylase (ATP:sulfate adenylyltransferase; EC 2.7.7.4), the first enzyme of the two-step sulfate activation sequence, was purified extensively from rat liver cytosol. The enzyme has a native molecular mass of 122 ± 12 kDa and appears to be composed of identical 62 ± 6-kDa subunits. At 30 °C and pH 8.0 (50 m m Tris-Cl buffer containing 5 m m excess Mg 2+), the best preparations have “forward reaction” specific activities of about 20 and 2 units × mg protein −1 with MoO 4 2− and SO 4 2−, respectively. The reverse (ATP synthesis) specific activity is about the same as the forward molybdolysis activity. The kinetic constants under the above conditions are as follows: KmA = 0.21 mM, K ia = 0.87 mM, K mB = 0.18 mM, K mQ = 0.65 μM, K iq = 0.11 μM, and K mP = 5.0 μM where A = MgATP, B = SO 4 2−, Q = APS, and P = total PP iat 5 mM Mg 2+. PP i is a mixed-type inhibitor with respect to MgATP and SO 4 2−. SeO 4 2− is an alternative inorganic substrate with a V max about 20% that of SO 4 2−. The product, APSe, is unstable. But in the presence of a sufficient excess of APS kinase, APSe is completely converted to PAPSe. The rate constant for nonenzymatic PAPSe hydrolysis was determined from measurements of the final steady-state reaction rate in the presence of limiting initial SeO 4 2− and a large excess of MgATP, ATP sulfurylase, APS kinase, and the other coupling enzymes and their cosubstrates. The results yielded a k of 2.4 ± 0.5 × 10 −3 sec −1 ( t 1 2 ca. 5 min). Phosphate is an effective buffer for enzyme purification and storage but inhibits catalytic activity, particularly at low substrate concentrations. In the presence of buffer levels of P i, the MgATP reciprocal plot of the SO 4 2−2-dependent reaction is concave-up. Inorganic monovalent oxyanions are dead end inhibitors competitive with SO 4 2−2 and apparently uncompetitive with respect to MgATP. The relative potencies are in the order ClO 3 2− > ClO 4 − > FSO 3 − > NO 3 −. Thiosulfate is also competitive with SO 4 2−2 but noncompetitive with respect to MgATP. Several divalent oxyanions (MoO 4 2−2, WO 4 2−2, CrO 4 2−2, and HAsO 4 2−2) promote the enzyme-catalyzed cleavage of MgATP to AMP and Mg PP i. The ratio V maxf K mA ranged from 0.7 to 200 for various reactive inorganic substrates. The cumulative results suggest the random binding of MgATP and the inorganic substrate but the ordered release of Mg PP rmi before APS. However, the relative contributions of the E → EA → EAB and E → EB → EAB paths cannot be evaluated. Chemical modification and inactivation protection studies suggest the presence of functionally essential argininyl, lysyl, and histidinyl residues.