A human hepatoma cell line, Hep G2, which synthesizes and secretes several components of the fibrinolytic system, was examined for the capacity to produce plasminogen activator (PA). PA activity was found to accumulate in medium conditioned by the cells but was not detected in cell extracts. Analysis of Hep G2 conditioned medium by SDS-polyacrylamide gel electrophoresis revealed multiple PAs of Mr 100,000, 60,000, and 52,000 daltons. In addition, a heterogeneous band of activity was present between the 100,000 and 60,000 dalton PAs. All detectable PA activity in conditioned medium fractionated at a single position after isoelectric focusing. The isoelectric point of this material (pI 8.6) was identical to that of purified urokinase. Anti-urokinase IgG, but not anti-tissue activator IgG, neutralized the activity of these PAs. Treatment of conditioned medium with 25 mM diisopropylfluorophosphate for 5 hr at 37 degrees C inactivated the 52,000 and 60,000 dalton PAs but had no effect on the 100,000 dalton PA or the heterogeneous zone of activity. The possibility that the absence of detectable PA activity in the cell extracts resulted from the presence of fibrinolytic inhibitors was considered. Addition of cell extract to purified urokinase resulted in a concentration-dependent reduction of urokinase-mediated fibrinolytic activity, consistent with the presence of such inhibitors. Acidification of the cell extracts to inactivate the inhibitor(s) revealed the presence of a latent, cell-associated PA activity. Thus, in addition to the other major components of the fibrinolytic system, Hep G2 cells produce multiple forms of a urokinase-like PA. Detection of these PAs may be obscured by the presence of cellular inhibitors.