The effects of variations in cell density on the expression of the plasminogen activator activity of a tumorigenic rat cell line were analyzed. At low cell densities, the plasminogen activator activity per cell was high and independent of cell density. As the cell density increased, the plasminogen activator activity per cell decreased until it eventually became inversely proportional to cell density. Inhibition of the plasminogen activator activity per cell by increases in cell density was not the result of the presence of a soluble inhibitor but seemed to require cell-to-cell contact. The Vmax per cell for the activation of plasminogen changed at high cell densities, but the Km did not change. This change in the Vmax per cell was in part the result of a change in the catalytic rate constant for the conversion of plasminogen to plasmin. This was inferred from studies on the kinetics of inhibition of plasminogen activator activity by diisopropyl fluorophosphate as a function of cell density. For cells growing at high densities, the rate of inhibition was constant, exhibiting a second-order rate constant of 2.6 × 10−2M−1 s−1. For cells growing at low densities, the plasminogen activator activity was inhibited at two different rates, one exhibiting a second-order rate constant of 2.6 × 10−2M−1 s−1 and the other exhibiting a second-order rate constant of 9.4 × 10−2M−1 s−1. We discuss the importance of cell density in assays of the plasminogen activator activity of cells, the use of this cell line to study the biochemical basis of the density dependence of plasminogen activator activity, and the density-dependent role of plasminogen activator activity in tumor formation and metastasis.