There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER)␣ or ER. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17-estradiol. One important system related to the activity of ER␣ and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82-101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ER␣ and ANG II type 1 and 2 (AT 1 and AT2) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca 2ϩ kinetics and the effect of 17-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca 2ϩ concentration measurements, immuno-and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca 2ϩ concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT2 receptors associate with Cav-1. Our results show that there is a close association of AT1, AT2, and ER␣ with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling. estrogen receptors; angiotensin type 1 and 2 receptors; phosphatidylinositol 3-kinase; intracellular signaling; tissue culture; angiotensin receptors CAVEOLAE are electron microscopy-identifiable plasma membrane invaginations (12,20,27). They are mainly formed in enriched cholesterol, sphingolipid, and glycosylphosphatidylinositol zones of the plasma membrane. The main structural protein of caveolae is caveolin (Cav); there are three isoforms expressed in mammal cells (Cav-1, Cav-2, and Cav-3). Adipocytes, endothelial cells, pneumocytes, and fibroblasts have the highest levels of Cav-1 and Cav-2, whereas Cav-3 expression is limited to muscle cell types [cardiac, skeletal, and smooth muscle cells (SMCs)] (28, 37).It has been postulated that Cav-1 acts also as a scaffold protein for signaling proteins. These proteins include G protein-coupled receptors (GPCRs), heterotrimeric G proteins, receptor tyrosine kinases, components of the renin-angiotensin system (RAS)-MAPK pathway, Src family tyrosine kinases, PKC isoforms, nitric oxide (NO) synthase, epidermal cell growth factor receptor (ECGF-R), and related receptor tyrosine kinases (8,1...