Blood components such as oxyhemoglobin are believed to cause cerebral vasospasm by inducing contraction and cell death in cerebral arteries. We have observed previously that oxyhemoglobin produces apoptotic changes in cultured endothelial cells. This study was undertaken to explore if bilirubin, a bi-product of hemoglobin degradation, will produce similar cytotoxicity in endothelial cells. Cultured bovine brain microvascular endothelial cells were incubated in four concentrations of bilirubin (10, 25, 50, and 100 microM) for varying times (6, 12, and 24 h). Control cells were incubated in saline or vehicle (NaOH solution, <0.01% of 0.01 N) for similar time periods. The cultured cells were then observed microscopically for evidence of cellular alterations. Bilirubin (10-100 microM) produced apoptosis that appeared time-dependent but not clearly concentration-dependent. Biochemical markers for apoptosis such as DNA fragmentation and PARP cleavage were induced by bilirubin. We conclude that endothelial cells may undergo apoptosis after exposure to bilirubin.