The release of plasminogen activator (PA) by corneal epithelium was studied utilizing pure culture of rabbit corneal epithelial cells and a sensitive photometric assay for the enzyme. The activity of PA measured in conditioned medium collected from the cultured cells was plasminogen-dependent, acid-stable, and free of interference by endogenous PA inhibitors. When serum was included in the culture medium, it was necessary to acid-treat the conditioned medium before PA assay in order to inactivate plasma-derived inhibitors. During a month of culture in serum-free medium, the cells released a low basal level of PA in the first week of growth and confluency. During the second week the cells progressively increased PA secretion at the onset of cell differentiation, reaching a peak (18-fold increase) in the third week when focal multilayering of cells occurred. Thereafter, the cells declined release, concomitant with globular aggregation of cells. A delay in the elevated release of PA was observed when the confluent phase was extended to 3 weeks, after which a substantial rise in PA level occurred simultaneously with the onset of multilayering. An in vitro model of corneal wound closure was established on confluent epithelial cells (grown in serum-containing culture medium) by mechanically removing 35-40% of cells in a central circular area. Complete wound closure was effected within 2-4 days by cell migration. The release of PA during the first day of wound closure was four times that of unwounded culture. The results indicated that PA secretion by corneal epithelium was associated with cell differentiation and cytomobility, processes that occur during stratification or wound closure. This conclusion is discussed with respect to a previously hypothesized role of PA in corneal injury and ulceration.