The degradation of heparan sulfate proteoglycan (HSPG) in basement membranes (BM) has been previously suggested to be accomplished by an endoglycosidase activity called heparanase which has not been isolated outside of platelets. HSPG degradation by heparanase has been associated with tumor cell invasion, angiogenesis, and growth factor function. In this study, we identify heparanase activity biochemically and immunologically in malignant human prostate carcinoma cells (PC-3M), linking platelet heparanase probes with the tumor heparanase activity observed. Concentrated conditioned medium from PC-3M cells was analyzed by a heparin-Sepharose affinity column. Three peaks eluted with 0.15, 0.35, and 0.5 M NaCl. Each peak was analyzed by incubation with 3H-labeled heparin as well as [3H]HSPG from EHS tumor BM. The 0.5 M peak material degraded [3H]-heparin by 17.2%, with little additional degradation by the other peaks in comparison to the conditioned medium from which they were obtained. Likewise, the same amount of the 0.5 M peak accounted for the majority of degradation (30.8%) of 3H-labeled HSPG. Interestingly, for the same amount of 0.5 M peak material, significantly more HSPG was degraded than heparin under the same conditions. In addition, carrageenan-lambda, an inhibitor of glycanase, completely inhibited the degradation of heparin and heparan sulfate proteoglycan by the 0.5 M peak. Using antibody to the N-terminus domain of platelet heparanase, a 60-kDa protein was identified by immunoblot in 0.5 M peak material. Additionally, immunohistochemical staining of human prostate carcinoma specimens showed granular staining at or near the cell membrane and near the luminal surface using antibody to the N-terminus and C-terminus domains of platelet heparanase. In summary, human prostate carcinoma cells show heparanase activity in conditioned medium that degrades heparin and BM HSPG and is detected by antibody to platelet heparanase. In addition, the membrane-associated staining in tissue sections of prostate cancer strongly correlates with the biochemical and immunological detection in conditioned medium of human PC-3M cells.