AbstractTo evaluate the proteolytic activities of oncogene-transfected 3T3 cells, we have developed a copolymerized substrate electrophoretic assay that permits the detection ofpicogram quantities of proteases produced by cells in culture. Our assay involves a gelatin substrate copolymerized in a polyacrylamide gel. Purified cell membrane preparations were run on gels and various protease activities were detected by amido black. Ras-transfected 3T3 cells appear to have a soluble metal-loprotease that may be transiently membrane bound and responsible for destruction of red blood cells (RBC). Oncogene-transfected NIH-3T3 cells have been demonstrated to have RBC cytolytic activity. We have previously shown that v-src-transfected 3T3 cells and their cell membranes cause RBC cytolysis which is inhibited by the protease inhibitor leupeptin. Here we report that both H-ras- and K-ras-transfected 3T3 cells and their cell membranes are cytolytic for RBC, but are inhibited by the metalloprotease inhibitor ethylene diamine tetraacetic acid. Using the gelatin substrate gel assay, we determined that some of the proteases were intrinsic to the oncogene expressing cells, while other proteases were secreted into the culture growth medium.