Estrogen has both rapid and longer-term direct effects on cardiovascular tissues mediated by the two estrogen receptors, ERα and ERβ. Previous work identified that estrogen regulates the expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMC). ERβ knockout mice have vascular dysfunction due to dysregulation of iNOS expression and these mice are hypertensive (Zhu et al, Science 2002;295:505-508). Here we report studies to examine the differential regulation of iNOS gene expression by ERα and ERβ. Immunoblotting and RT-PCR studies revealed that different VSMC lines expressed different levels of ERα and ERβ protein and mRNA. VSMC from different vascular beds were studied, including aortic VSMC expressing ERα and radial VSMC expressing ERβ. E2 inhibited NO production and iNOS protein expression in aortic VSMC. Human iNOS promoter reporter studies revealed suppression of iNOS reporter activity by E2 in aortic VSMC, and stimulation of iNOS reporter activity by E2 in radial arterial VSMC. In heterologous expression studies of COS-7 cells lacking endogenous ER, E2 treatment of COS-7 cells did not alter iNOS reporter activity in the presence of ERα, while reporter activity increased 2.3 fold in the presence of ERβ. Similar experiments in COS-7 cells using the selective estrogen receptor modulator raloxifene showed that raloxifene caused a reduction in iNOS reporter activity with ERα co-expression, and an increase with ERβ co-expression. Rat VSMC expressing ERβ but not ERα also showed increased iNOS reporter activity with E2 treatment, an effect lost when ERα was introduced into the cells. Taken together, these data support that hiNOS transcription is positively regulated by ERβ and negatively regulated by ERα in VSMC, supporting differential actions of these two estrogen receptors on a physiologically relevant gene in vascular smooth muscle cells.