The role of caveolins, signature proteins of caveolae, in arterial Ca²⁺ regulation is unknown. We investigated modulation of Ca²⁺ homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca²⁺ transients were simultaneously measured using voltage-clamped single cells. Membrane depolarization triggered Ca²⁺ current and increased intracellular Ca²⁺ concentration ([Ca²⁺]_i). Upon repolarization, elevated [Ca²⁺]_i returned to the resting level. Ca²⁺ removal rate was determined from the declining phase of the Ca²⁺ transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to the amino acid residues 82-101 of caveolin-1, significantly slowed Ca²⁺ removal rate at a measured [Ca²⁺]_i of 250 nM with little effect at a measured [Ca2+]i of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to the amino acid residues 55-74 of caveolin-3, also significantly slowed Ca²⁺ removal rate at a measured [Ca²⁺]_i of 250 nM with little effect at a measured [Ca²⁺]_i of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2 related inhibitory peptide and cyclosporine A, inhibitors for calmodulin, Ca²⁺-calmodulin-dependent protein kinase II and calcineurin, respectively, also significantly inhibited Ca²⁺ removal rate at a measured [Ca²⁺]_i of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca²⁺ ATPase inhibitor, also significantly inhibited Ca²⁺ removal rate at a measured [Ca²⁺]_i of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca²⁺ removal of resistance artery smooth muscle cells.