Choline sulphokinase, free of endogenous choline, APS kinase and PAPS-degrading enzymes, but containing some ATP sulphurylase, was prepared from Aspergillus nidulans. The pH optimum of the enzyme was 7.8 and maximum stability was in the region pH 8.5–10.5. Neither Mg2+ nor other metal ions, were essential for activity. Inhibition by sulphydryl reagents suggested the necessity for free SH groups in the enzyme. Only 3′-phosphoadenosine-5′-phosphosulphate (PAPS) could act as the sulphate donor and of 49 compounds tested, including many choline analogues, only three, choline, dimethylethylaminoethanol and dimethylaminoethanol could act as sulphate acceptors; the Km values being 12, 20 and 25 mM respectively. The binding sites on the enzyme for PAPS and choline appear to be independent of one another. A number of compounds were found to be inhibitors of the reaction the most potent of which was hexadecyltrimethylammonium bromide. The inhibition by p-nitrophenol, carnitine and dimethylaminopropanel-ol was competitive. The transfer of sulphate from PAPS to choline by choline sulphokinase appears to be irreversible, at least in vitro.