We investigated the importance of the urokinase (uPA)-plasmin system in fostering invasion of human lung cancer cells through artificial basement membranes composed of Matrigel. Eight cell lines (including 1 small cell and 7 non-small cell lines) were examined. One cell line did not express any components of the urokinase system. Four cell lines had substantial levels of endogenous uPA detectable on their surfaces. Three of these cell lines co-expressed the plasminogen activator inhibitor PAI-1 in addition to uPA. Assays for invasiveness revealed 4 cell lines capable of traversing a Matrigel barrier, including the 3 which co-expressed uPA, PAI-1 and uPA receptor. Surprisingly, the cell line expressing only uPA and uPA receptor displayed no invasive capacity despite levels of secreted uPA more than 20-fold higher than the other cell lines studied. Based on these observations, we hypothesized that both uPA and PAI-1 might be important for invasion by lung tumor cells, at least in vitro. We therefore tested polyclonal antibodies which inhibit uPA and PAI-1 activity for their effects on the highly invasive H292 cell line. After 3 days, invasive capacity was inhibited by antibodies to both uPA and PAI-1 in a dose-dependent manner. The plasmin inhibitor aprotinin reduced H292 cell invasion by 70%. Taken together, our data demonstrate that in cultured human lung cancer cells the uPA-plasmin system is important in promoting invasion into basement membranes and suggest that a critical balance between uPA and PAI-1 is necessary for optimal invasiveness. Our data are consistent with results from recent clinical studies showing that PAI-1 expression in tumor tissue is an adverse prognostic feature.