Baseline cellular plasminogen activator (PA) activity, the cellular proteins responsible for variations in PA activity and the effect of cellular PA activity on cellular adherence to and lysis of fibrin substrate were evaluated in three human transitional carcinoma cell lines. Net PA activity in the cell lines 253J, 639V and 647V was measured using a chromogenic substrate assay. These lines were then analyzed to determine the specific protein(s) responsible for differences in PA activity. mRNA and protein levels of cellular uPA, tPA, PAI1 and PAI2 were measured using Northern blot analysis and ELISA assays. Intact cells were used in an in vitro fibrinolysis assay so as to correlate cell biology with protein and mRNA level observations. Net cellular PA activity in the three cell lines varied over a 20-fold range (253J > 639V > 647V). Net PA activity demonstrated a direct correlation with mRNA transcript and protein levels of uPA/low levels of tPA mRNA were detected in the 253J line. However, tPA protein was not detectable in any of the lines. Both PAI1 and PAI2 were detected in varying amounts in each of the three cell lines. In vitro assays demonstrated a direct correlation between net PA activity and plasminogen dependent fibrin substrate lysis. Cellular adherence to fibrin varied as an inverse function of net PA activity. These findings suggest that variations in cellular uPA levels are principally responsible for variations in PA activity between cell lines. Variations in net PA activity are in turn reflected at the cellular level by differences in ECMP substrate lysis and cellular adherence to fibrin.