The interaction of high density lipoproteins (HDL) with the HDL receptor stimulates the translocation of cholesterol from intracellular pools to the plasma membrane where the cholesterol becomes available for removal by appropriate acceptors. The role of signal transduction through protein kinase C in HDL receptor-dependent cholesterol translocation and efflux was examined using cholesterol-loaded cultured human skin fibroblasts. Treatment of cells with HDL3 activated protein kinase C, demonstrated by a transient increase in membrane associated kinase activity. Kinase activation appeared to be dependent on binding of HDL3 to the HDL receptor, since tetranitromethane-modified HDL3, which does not bind to the receptor, was without effect. Translocation of intracellular sterol to the plasma membrane was stimulated by treatment of cells with the protein kinase C activators, dioctanoylglycerol and phorbol myristic acetate, and the calcium ionophore A23187. Conversely, treatment of cells with sphingosine, a protein kinase C inhibitor, reduced HDL3-mediated translocation and efflux of intracellular sterols. However, sphingosine had no effect on efflux of labeled cholesterol derived from the plasma membrane. Down-regulation of cellular protein kinase C activity by long term incubation with phorbol esters also inhibited HDL3-mediated efflux of intracellular sterols and abolished the ability of sphingosine to further inhibit HDL3-mediated efflux. These studies support the conclusion that HDL receptor-mediated translocation and efflux of intracellular cholesterol occurs through activation of protein kinase C.