Secretion of plasminogen activators and their inhibitors was examined in cultures of human retinal pigment epithelial (RPE) cells. The methods employed were zymography and reverse zymography, solid-phase immunocapture assay, metabolic labeling followed by immunoprecipitation, and immunofluorescence. The results showed that these cells produce urokinase-type plasminogen activator (u-PA) and a plasminogen activator inhibitor (PAI) which is immunologically and biochemically similar to PAI-1. Tissue-type plasminogen activator activity (t-PA) was not detected, but we detected small amounts of t-PA in an inactive complex with inhibitor in RPE cell-conditioned media. We conclude that RPE cells have the potential to utilize u-PA-catalyzed plasminogen activation which is subject to regulation by PAI-1. These results may have a bearing on the pathogenesis of proliferative retinal diseases.