Changes of the cell surface proteolytic activity during the cell cycle in vitro were reported. Using an easy assay, with casein as a substrate, the proteolytic activity on the surface of Ehrlich ascites tumor (EAT) cells grown in vivo was determined. The cleavage of casein incubated with EAT cells increased linearly for 20 min and permitted reproducible enzyme activity determinations. If the proliferation of exponentially growing EAT cells was partially synchronized by an intraperitoneal bleomycin injection, a significant increase of the surface enzymatic activity was observed in cells with an increased DNA content. This finding supports the results obtained with transformed cells in vitro, indicating that elevated proteolytic surface activity occurs in the late synthesis phase and prior to mitosis. However, the observed effect may also be due to changes of gene expression caused by bleomycin.