Plasminogen activator (PA) activity was studied in a transplantable murine mammary adenocarcinoma (M3), moderately metastatic to lungs, and in a highly metastatic variant tumor (MM3) to establish whether a correlation existed between this enzyme and the tumors' metastasizing abilities. The cell-associated and secreted PA activities from primary cultures of both tumors, as well as from solid tumor homogenates, were quantitated by means of a fibrinolytic assay. Immunoneutralization and zymographic assays were done to identify the PA present in both tumors. In culture, the highly metastasizing MM3 cells produced (secreted plus cell-associated activator) 3.3-fold higher PA levels than the M3 cells. This difference was mainly attributed to the enhanced secreting ability of MM3, as these tumor cells secreted 102 times their cell-associated PA activity within 24 hr, while M3 cells secreted only 11 times this activity during the same period. PA activity was also significantly higher in MM3 than in M3 tumor homogenates. Acid-labile inhibitors and non-specific proteases were not detected. In both tumors, PA was characterized as urokinase-type, with a molecular weight of approximately 48 kDa. These results suggest that, in this model, PA plays a role in metastasis formation.