Endogenous cysteine proteinase inhibitors (CPIs) presumably regulate lysosomal cysteine endopeptidases (CPs), such as cathepsins B and L, in vivo. An imbalance between CPs and CPIs in carcinomas, possibly due to impaired inhibition of proteinases, was reported. Ovarian carcinoma contain high levels of Stefin B and about twentyfold less Stefin A compared to normal epithelial tissue. Stefin B was isolated and characterized. We used alkaline treatment, affinity chromatography on Cm-papain Sepharose, followed by gel filtration and ion-exchange chromatography and anti-Stefin B-Sepharose 4B to isolate two major isoforms of Stefin B with pI values 5.9 and 6.5. M(r) of ovarian Stefin B was close to 14,000 as judged by SDS-PAGE and had a blocked N-terminus. It strongly inhibited papain (Ki = 0.11 nM) and cathepsin L (Ki = 0.035 nM), but only moderately cathepsin B (Ki = 130 nM). As these properties are similar to Stefin B from human and bovine origin, as well as to Stefin B from human histiosarcoma, we believe that tumor Stefin B does not differ from normal Stefin B.