Three cysteine proteinases were isolated from the post-myofibrillar fraction of rat skeletal muscle. Proteinase I preferentially hydrolyzes Z-Phe-Arg-NMec with pH optimum at 8-9. The enzyme activity is stabilized by ATP against thermal inactivation. Proteinase II and III were not resolved by anion-exchange chromatography, by affinity chromatography on Arginine-Sepharose or by gel filtration. Proteinase II, splitting Bz-Val-Gly-Arg-NMec optimally at pH 10-10.5, is inactivated by ATP, whereas Proteinase III, hydrolyzing Suc-Ala-Phe-NMec at pH 7-7.5 is not affected by the nucleotide. The molecular mass of proteinase I is about 750 000 and that of proteinase II and III is about 650 000, as determined by gel filtration.