Studies investigating the mechanisms that govern the expression of the human angiotensin II (Ang II) type 1 receptor (hAT1R) gene have progressed slowly due to the lack of human cell lines that express the AT1R. Recently, however, an immortalized human trophoblast cell line (HTR-8/SVNeo) was demonstrated to respond to Ang II. Therefore, we utilized this cell line to characterize the AT1R expressed on the cell surface and to investigate the mechanisms by which the hAT1R gene is regulated in these cells. HTR-8/SVNeo cells were shown to express functional high affinity AT1Rs having a Bmax value of 114+/-11 fmol/mg protein and a Kd value of 0.14+/-0.1 nM. Additionally, Ang II-induced IP3 production was mediated via the AT1R. Deletional analysis of the hAT1R promoter localized a major basal regulatory sequence within the -105 to -79 bp region, relative to the transcription start site, in HTR-8/SVNeo cells. Electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay demonstrated that the transcription factors, Sp1 and Sp3, interact with this region of the hAT1R promoter in vitro and in vivo. Taken together, our data demonstrate that HTR-8/SVNeo cells express functional AT1Rs and that basal level expression of this gene is regulated, in part, by Sp1 and Sp3 in this cell line.