We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.