The synthesis, storage and regulated secretion (acute release) of tissue-type plasminogen activator (tPA) were studied in cultured rat heart endothelial (RHE) cells. In this study, special attention was paid to the correspondence between tPA metabolism in RHE cells in vitro and tPA metabolism in rats in vivo. RHE cells synthetized tPA, as shown by a spectrophotometric activity assay, by fibrin autography and by an ELISA for tPA antigen. The synthesis of tPA was strongly enhanced by phorbol myristate acetate, by all-trans-retinoic acid, by 8-bromoadenosine 3':5' cyclic- monophosphate (8-br-cAMP) and by compounds that enhance cAMP synthesis, such as cholera toxin and forskolin. Urokinase-type plasminogen activator and plasminogen activator inhibitor were not detected. tPA was constitutively secreted by RHE cells, but was also present in an intracellular pool of small dense granules, from which it could be acutely released by stimulating the cells with thrombin or the calcium ionophore A-23187. This release was maximal during the first minutes after stimulation. In all these aspects of tPA metabolism, RHE cells closely resembled rat endothelial cells in vivo. It is concluded that cultured RHE rat heart endothelial cells constitute a relevant in vitro model for studying endothelial tPA metabolism.