The irritating effect of parenterally administered antibiotics on vessels is a common clinical problem. In a previous study we found that solutions of three commonly used antibiotics, cefuroxime, erythromycin and dicloxacillin, exerted cytotoxic effects on endothelial cells after 24 hr exposure. In contrast benzylpenicillin did not have such effects. In the present study, endothelial cells of different origin were exposed to these four antibiotics at higher concentrations than in the previous investigation but only for 5, 30 and 60 min. Incorporation of 3H-thymidine in the cells as a measurement of DNA synthesis was used as cytotoxic assay. A concentration-dependent and time-related inhibition was found after exposure to erythromycin and dicloxacillin but not after exposure to cefuroxime and benzylpenicillin. The effects were similar on the three different cell types used in the experiments. This study demonstrates that the cytotoxic effects differ even after short-term exposure to the antibiotics. In contrast to the previous study, cefuroxime lacks cytotoxicity when endothelial cells are exposed for less than one hour. The short-term exposition model used in this study should be more predictive as it mimics in vivo conditions better.