Heparin, which is widely used as an anticoagulant, has been shown to have antiatherosclerotic and antihypertensive effects in animals and humans. These effects are mediated by the inhibition of endothelin-1 (ET-1) production in endothelial cells. To clarify the mechanism of this inhibition, we investigated the effect of heparin on transcriptional regulation of the ET-1 gene in bovine aortic endothelial cells (BAEC) cultured in fetal calf serum. ET-1 mRNA expression was significantly suppressed by heparin in a dose-dependent manner. Promoter analysis revealed that the minimum ET-1 promoter containing only the GATA and AP-1 sequences as positive cis-acting sites in the ET-1 promoter is sufficient for this suppression. Gel mobility shift assays using oligonucleotides encoding the ET-1 AP-1 and ET-1 GATA sites confirmed that both AP-1 and GATA binding activities in BAEC nuclear extract were markedly inhibited by heparin. Western blot analyses indicated that heparin completely blocked extracellular signal-regulated kinase (ERK) activation, and inhibiting ERK activity resulted in loss of heparin-dependent inhibition of the ET-1 gene. These data indicate that the ET-1 mRNA level is negatively regulated by heparin at the transcription level, through modification of AP-1 and GATA protein binding activities, which direct the ET-1 promoter in BAEC. This effect may be mediated, at least in part, through inhibition of ERK activity.