The class B, type I scavenger receptor (SR‐BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR‐BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin‐1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin‐1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester‐induced differentiated THP‐1 cells. The goal of the present study was to determine if the expression of caveolin‐1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J‐774 cell lines that stably expressed caveolin‐1. Transfection with caveolin‐1 cDNA did not alter the amount of ¹²⁵I‐labeled HDL that associated with the cells, although selective uptake of HDL [³H]cholesteryl ether was decreased by approximately 50%. The amount of [³H]cholesterol effluxed to HDL was not affected by caveolin‐1. To directly address whether caveolin‐1 inhibits SR‐BI‐dependent selective cholesteryl ester uptake, we overexpressed caveolin‐1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR‐BI. Caveolin‐1 inhibited the selective uptake of HDL [³H]cholesteryl ether by 50–60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR‐BI to demonstrate that the increase in selective [³H]cholesteryl ether uptake previously seen in differentiated THP‐1 cells was independent of SR‐BI. Finally, we used β‐cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR‐BI‐dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin‐1 is a novel negative regulator of SR‐BI‐dependent selective cholesteryl ester uptake.