High ambient glucose concentration, linked to vascular complications in diabetes in vivo, modulates mRNA expression of fibronectin, collagen, tissue-type plasminogen activator, and plasminogen activator inhibitor and induces delayed replication and excess cell death in cultured vascular endothelial cells. To determine the role of high ambient glucose (30 mmol/1) in apoptosis, paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were exposed to both high (30 mmol/1) and low (5 mmol/1) concentrations of glucose for short-term (24, 48, and 72 h) and long-term (13 ± 1 days) experiments. Incubation of HUVECs with high glucose for >48 h increased DNA fragmentation (13.7 ± 6.5% of total DNA, mean ± SD) versus cultures kept in 5 mmol/1 glucose (10.9 ± 5.6%, P < 0.005), as measured by [3H]thymidine assays. Data were confirmed by apoptosis-specific fluorescence-activated cell sorter analysis of confluent HUVEC cultures, which displayed after long-term exposure to 30 mmol/1 glucose a 1.5-fold higher prevalence of apoptosis than control cultures exposed to 5 mmol/1 glucose (P < 0.005). In contrast, no increase in DNA fragmentation in response to 30 mmol/1 glucose was seen for standardized cell lines (K 562, P 815, YT) and fibroblasts. Expression of clusterin mRNA, originally reported to be a molecular marker of apoptosis, was only slightly affected by short-term (24-h) high-glucose exposure but was significantly reduced after long-term incubation in 30 mmol/1 glucose (82.2 ± 13.8% of control) versus 5 mmol/1 glucose, which questions the role of clusterin gene expression as a marker of apoptosis. The results demonstrate that high ambient glucose can promote apoptosis in HUVECs in vitro and suggest potential endothelial damage by hyperglycemia in diabetic patients.