We have identified and characterized 4 probands with severe familial HDL deficiency (FHD) characterized by a lack of mature, α-migrating LpAl HDL particles. Despite thorough clinical, biochemical and molecular characterization, no known cause of HDL deficiency has been identified in these families. We examined the possibility that a cellular defect may cause an impaired HDL-mediated cholesterol efflux in FHD. Fibroblasts were obtained from skin biopsies of normal and FHD subjects. Using cholesterol-loaded cells, selective radiolabeling of cellular cholesterol pools were used for efflux studies. First, confluent growth-arrested cells were labeled with [ 3 H]-cholesterol to specifically enrich plasma membrane as established by sucrose density gradient ultracentrifugation; most of the [ 3 H]-cholesterol (80-95%) was found within the plasma membrane. In these conditions, normal HDL 3 (1 mg/mL of proteins) was able to desorb [ 3 H]-cholesterol from normal and FHD cells in a time dependent (up to 24 hours), linear kinetic fashion, with no difference in rates of efflux between FHD and normal cells. Second, to introduce a high fraction of label into intracellular compartments, cells were labeled with [ 3 H]-cholesterol during growth. Under these conditions, apo Al or HDL 3 (100 μg/mL of proteins) had a markedly reduced effect in promoting radiolabeled cholesterol efflux in FHD cells (25-30%) compared with cells from normal subjects. When using [ 3 H]-mevalonolactone to label cells, significantly less newly synthesized sterols are found in the plasma membrane of FHD cells, compared with normal cells ( 2-3-fold). This data show that intracellular cholesterol efflux is abnormal in FHD fibroblasts and suggest a defect in cellular trafficking of cholesterol to the cell membrane. This would lead to the formation of small, lipid-depleted nascent HDL particles (preβ mobility LpAI particles) that are rapidly cleared from plasma.