Endothelial cells from both human retinal microvessels (HME) and fetal bovine aortic endothelium (FBAE) were grown in aggregate cultures alone, or with either retinal pigment epithelium (RPE) cells or fibroblasts. The levels of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in the conditioned media of the various aggregate types were measured. High PA levels were detected in the conditioned medium of pure endothelial cell aggregates (equal to 140% and 124% of urokinase control for HME and FBAE, respectively), and high PAI levels were associated with pure RPE aggregates (inhibiting 93% of the urokinase control). The conditioned medium of pure fibroblast aggregates had very low levels of either PA or PAI. When RPE cells were aggregated with FBAE or HME cells into mixed (heterogenous) aggregates, the PA measured in the conditioned medium was equal to 22% and 30% of the urokinase control, respectively. The PA level in the conditioned medium of mixed fibroblast-FBAE cell aggregates was higher, 104% of the control, and the difference was statistically significant (P less than 0.001). Co-incubation of pure RPE aggregates with pure FBAE aggregates or with pure HME aggregates resulted in PA activity in the conditioned medium that was equal to 110% and 96% of the control, respectively. The PA level found when pure FBAE cell aggregates were co-incubated with pure fibroblast aggregates was higher, 134% of the control, and the difference was statistically significant (P less than 0.001). Our results indicate that RPE cells can reduce endothelial cell PA, probably through both direct contact between the cells and PAI production. Fibroblasts did not have this influence on endothelial cell PA.