The sulfate-activation pathway consists of the sequential action of ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) and adenosine 5'-phosphosulfate kinase ATP:adenylylsulfate 3'-phosphotransferase, EC 2.7.1.25). Both sulfurylase and kinase from rat chondrosarcoma were copurified through substrate affinity chromatography using stable analogs of APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). A 56-kDa protein, containing both activities, was then purified to apparent homogeneity through reversed-phase chromatography and observed on denaturing polyacrylamide gels. The molecular mass of the native active unit containing both activities in the purified preparation corresponded to approximately 60 kDa by analytical gel filtration. Coincident binding and elution of ATP sulfurylase and APS kinase by immunoaffinity chromatography, using polyclonal serum generated against the 56-kDa protein, also demonstrated that the enzyme contains both activities. Lastly, a single N-terminal amino acid sequence was obtained from the 56-kDa band isolated by gel electrophoresis. These results all suggest that ATP sulfurylase and APS kinase from rat chondrosarcoma residue on a single bifunctional protein.