Endothelial cells are a major source of endothelin-1 (ET-1), but the possibility that vascular adventitial fibroblasts (AFB) generate ET-1 has not been explored. We hypothesized that aortic AFB have the ability to produce ET-1, which may contribute to extra cellular matrix (ECM) synthesis. Vascular AFB were isolated from mouse aorta and incubated with various concentrations of angiotensin II (Ang II). mRNA levels of preproET-1 and type I procollagen I were detected by RT-PCR. ET-1 levels in the culture medium were measured by ELISA. Protein levels of procollagen I were detected by western blot. Ang II (10nM, 100nM and 1µM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (n=4, p<0.05). The induction of preproET-1 mRNA was accompanied by the release of the immunoreactive peptide ET-1 (n=6, p<0.05). The Ang II evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100µM), an AT₁-receptor antagonist but not PD123319 (100µM), an AT₂-receptor antagonist. To further confirm our findings, AFB preproET-1 was cloned and then sequenced bi-directionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (Accession: AB081657). Moreover, Ang II induced type I procollagen mRNA and protein expression was inhibited by BQ123 (10µM), an ET_A-receptor inhibitor, but not BQ788 (10µM), an ET_B-receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of ECM synthesis. The results demonstrate that vascular AFB are able to synthesize and release ET-1 in response to Ang II.