Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells. PMCA4 is the predominant isoform operating in conditions of high intracellular calcium during contraction. PMCA appears to be localized in lipid rafts and caveolae. In this study we examined the effects of the PMCA4-selective inhibitor caloxin 1c2 (5 microM) in intestine of caveolin-1 knockout mice and in bovine tracheal smooth muscle after caveolae disruption on PMCA4 function. Small intestinal tissues from control mice treated with caloxin 1c2 showed a higher contractile response of the longitudinal smooth muscle to Carbachol (10 microM) when compared to control tissues treated with a similar concentration of a control peptide. This effect of caloxin 1c2 was not found in tissues from caveolin-1 knockout mice. Immunohistochemistry and Western blotting of membrane fractions showed that PMCA was co-localized with caveolin-1 in smooth muscle plasma membrane in control tissues. One of the PMCA4 splice variant bands was missing in the lipid raft-enriched fraction prepared from caveolin-1 knockout tissue. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin-1 and PMCA4b immunoreactivities, previously co-localized in the smooth muscle plasma membrane, and to the loss of the increase in Carbachol-induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.