Cysteine-proteinase activity was observed in homogenates of human-cadaver renal cortex. This activity co-purified with renin enzymic activity until separation by aminohexyl-Sepharose--pepstatin affinity chromatography. The cysteine proteinase was purified 1780-fold after the following successive chromatographic procedures: Sephadex G-75, DEAE-cellulose DE-52, and an organomercurial affinity resin. The proteinase activity was dependent upon activation by thiol-containing compounds such as dithiothreitol, as well as by EDTA, and was inhibited by the thiol-group-specific alkylating reagents iodoacetic acid and N-ethylmaleimide. DE-52 cellulose chromatography resolved the cysteine proteinase into two components. On the basis of molecular size (26 000 daltons), activity as a function of pH, stability as a function of pH, substrate specificity and thermal lability, the major component (95%) has been identified as cathepsin B. The DE-52 cellulose elution pattern of the minor component (5%) is suggestive of cathepsin H [Schwartz & Barrett (1980) Biochem. J. 191, 487-497] Enzymic activity was determined with synthetic substrates, in particular alpha-N-benzoyl-DL-arginine 2-naphthylamide (Bz-Arg-NNap), thus precluding the detection of cathepsin L [Kirschke, Langner, Wiederanders, Ansorge, Bohley & Broghammer (1976) Acta Biol. Med. Germ. 35, 285-299]. Inhibition by dimethyl sulphoxide was observed in the determination of Km = 7.0 +/- 0.4 mM for the substrate Bz-Arg-NNap, and care must therefore be taken in the preparation of substrate solutions.