Spontaneous mammary tumors from C3H/HeJ mice and transplants established from mammary tumors were investigated for their capacity to secrete thiol-dependent proteinase activity in organ culture explants. More activity was detected in culture media from spontaneous tumors than from transplanted spontaneous tumors. The accumulation of thiol proteinase in the culture medium was inhibited by cycloheximide, hydrocortisone, and aldosterone, but not by estradiol or the peptide hormones insulin or prolactin. The thiol proteinase is similar in enzymic properties to lysosomal cathepsin B, but its physical properties are different. It is stable to alkaline pH, has a larger molecular size on gel filtration (relative M.W. 39,000) and shows a different isoenzyme pattern to liver cathepsin B on analytical isoelectric focusing. The characteristics of this thiol proteinase are very similar to an enzyme secreted from malignant human breast tumors.