Endothelin-1 (ET-1) is a potent vasoconstrictor and growth-promoting mediator that is involved in the maintenance of vascular tone within the healthy circulation. However, a pathogenic role has been implicated by its overproduction in a number of cardiovascular diseases, which include pulmonary hypertension, congestive heart failure, atherosclerosis, and coronary vasospasm. ET-1 mRNA expression and peptide production in human vascular smooth muscle cells (HVSMCs) are markedly increased by exposure to tumor necrosis factor-α and interferon-γ. The intracellular signaling mechanism involved in this pathway is not known. Because the transcription factors nuclear factor-κB (NF-κB), signal transducer and activator of transcription 1 (STAT1), and interferon regulatory factor-1 (IRF-1) often mediate the effects of cytokines in target cells the aim of this study was to determine whether the production of ET-1 after exposure of HVSMCs to cytokines depends upon synergism between NF-κB and STAT1/IRF-1. Immunoblotting showed that cytokine-stimulation of ET-1 release in VSMCs involves nuclear translocation of NF-κB and STAT1. Cytokines also induced an increase in IRF-1 protein expression. Antisense oligonucleotides to NF-κB, STAT1, and IRF-1 significantly inhibited cytokine induced ET-1 release. In conclusion, NF-κB, STAT1, and IRF-1 activation are involved in the stimulation by cytokines of ET-1 release from HVSMCs. However, nuclear run-on assays would provide definitive proof that ET-1 is regulated transcriptionally by cytokines. Because up-regulated production of ET-1 within VSMCs may underlie the causative role of ET-1 in a number of disease states, this finding indicates that NF-κB, STAT1, and IRF-1 within HVSMCs could be central to a number of vascular pathologies and that inhibition of this pathway could be of therapeutic benefit.