Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [I251]fibrin lysis assay, and high-and low-activity subclones were isolated; these retained high-or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.Plasminogen activator (PA) is a serine protease which activates plasminogen to plasmin, a fibrinolytic enzyme (21). Many different cell types produce PA; certain types of normal cells, cells transformed by chemicals or viruses, and tumor cells exhibit relatively high enzyme levels (7,14,21). Cell types that produce elevated levels of PA are generally invasive, which may be a direct result of their ability to generate protease activity and consequently to disrupt the surrounding tissue milieu (13).We have demonstrated that herpesvirustransformed cells activate plasminogen more efficiently than hamster embryo fibroblasts. These studies include cells transformed by herpes simplex virus types 1 or 2 (HSV-1, HSV-2) or cytomegalovirus (1, 9,24). Because hamster cell lines transformed by HSV-1 or HSV-2 also produce metastatic tumors when inoculated into syngeneic animals (5, 6), we wanted to assess the relationship between PA production and the tumorigenic potential of the herpesvirus-transformed cells. We selected a hamster cell line transformed by partially inactivated HSV-2 strain 333 for these experiments because we had utilized this cell line and virus strain previously (1, 5) and because it is known to produce metastatic lung nodules in an experimental hamster system (17).Other investigators have attempted to correlate PA activity with transformation and tumorigenicity in other animal systems...