There is evidence suggesting that the diabetic state adversely affects replication of certain cell populations. We document that exposure to high ambient glucose (20 mM) induces delay in various phases of the cell cycle of human endothelial cells in primary culture. Cells in S phase were labeled with bromodeoxyuridine (an analogue of thymidine), and the cell-cycle position of the labeled cohort was analyzed by flow cytometry at successive time points. The movement of cells exposed to high glucose for 7-8 days was retarded both in S and G2 phases so that the increase in bromodeoxyuridine-positive cells over 24 h was 1.6-fold, versus 2.0-fold in control cultures. In experiments in which mitotic arrest with vinblastine was used to investigate the movement of cells out of G1 phase without interference from reentering cells, depletion of the G1 compartment was significantly inhibited in cultures grown in high glucose. The effects of chronic high glucose on cell cycle occurred while total protein synthesis was not diminished. Acute exposure to high glucose had no effect on cell-cycle traversal or cell generation time. Cell-cycle abnormalities observed in this study may relate to the DNA damage we have previously observed in endothelial cells exposed to high glucose and, if occurring in vivo, could be of pathogenetic importance for the vascular lesions and teratogenicity of diabetes.