Cultures of Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF) produce 50-fold more of the protease plasminogen activator (PA), than do normal chick embryo fibroblasts. Treatment of RSVCEF cultures with the tumor promoter phorbol myristate acetate (PMA) further enhances (8- to 12-fold) the level of PA activity. Increased levels of PA activity in RSVCEF are observed as early as 1 to 2 hr after PMA treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates that the PA produced by PMA-treated cultures has a molecular weight identical to that of the PA produced by untreated cultures. PA induction by PMA cannot be accomplished in cell-free extracts, but requires protein synthesis in intact cells. Under serum-free conditions, PMA-treated RSVCEF secrete high levels of PA for 4 to 6 days and undergo pronounced morphological alterations. Modified culture conditions and the use of PMA to induce PA has allowed for the accumulation of large amounts of RSVCEF culture fluid and the subsequent purification of the enzyme. The sensitivity of transformed CEF to PMA and the generation of enhanced proteolytic activity in the cellular microenvironment may provide a model system to examine the role of both PA in malignant transformation and PMA in tumor promotion.