Mitomycin C (MMC) is a bifunctional alkylating agent used in cancer chemotherapy. MMC therapy occasionally results in pulmonary vascular injury, including alterations in endothelial cells. Reactive metabolites of MMC can cross-link DNA, and its cytotoxicity has been attributed in part to this capacity, but effects in vascular cells have not been explored extensively. Accordingly, the direct effects of MMC on cultured porcine pulmonary artery endothelial cells (PECs) were examined. A single administration of MMC (0-10 microM) to PEC monolayers resulted in concentration-dependent cytolytic injury that was delayed in onset and progressive in nature. Cells treated at subconfluent densities were inhibited in their ability to proliferate. MMC treatment resulted in DNA cross-linking at concentrations (0.01-1 microM) that inhibited cell proliferation but caused only limited overt cytotoxicity, supporting an association between DNA cross-linking and impairment of cell division. This pattern of PEC injury is reminiscent of that seen after treatment with another pneumotoxic, bifunctional alkylating agent, monocrotaline pyrrole. The similarity of the endothelial cell response to different bifunctional alkylating agents suggests that DNA cross-linking may inhibit cell proliferation and thereby limit the repair capacity of endothelial monolayers. This might contribute to the progressive pulmonary vascular injury that occurs after administration of certain DNA cross-linking agents in vivo.