—Familial high density lipoprotein (HDL) deficiency (FHD) is a genetic lipoprotein disorder characterized by a severe decrease in the plasma HDL cholesterol (-C) level (less than the fifth percentile). Unlike Tangier disease, FHD is transmitted as an autosomal dominant trait. FHD subjects have none of the clinical manifestations of Tangier disease (lymphoid tissue infiltration with cholesteryl esters and/or neurological manifestations). Plasmas from FHD subjects contain pre–β-migrating HDLs but are deficient in α-migrating HDLs. We hypothesized that a reduced HDL-C level in FHD is due to abnormal transport of cellular cholesterol to the plasma membrane, resulting in reduced cholesterol efflux onto nascent HDL particles, leading to lipid-depleted HDL particles that are rapidly catabolized. Cellular cholesterol metabolism was investigated in skin fibroblasts from FHD and control subjects. HDL₃- and apolipoprotein (apo) A-I–mediated cellular cholesterol and phosphatidylcholine efflux was examined by labeling cells with [³H]cholesterol and [³H]choline, respectively, during growth and cholesterol loading during growth arrest. FHD cells displayed an ≈25% reduction in HDL₃-mediated cellular cholesterol efflux and an ≈50% to 80% reduction in apoA-I–mediated cholesterol and phosphatidylcholine efflux compared with normal cells. Cellular cholesterol ester levels were decreased when cholesterol-labeled cells were incubated with HDL₃ in normal cells, but cholesterol ester mobilization was significantly reduced in FHD cells. HDL₃ binding to fibroblasts and the possible role of the HDL binding protein/vigilin in FHD were also investigated. No differences were observed in ¹²⁵I-HDL₃ binding to LDL-loaded cells between FHD and control cells. HDL binding protein/vigilin mRNA levels and its protein expression were constitutively expressed in FHD cells and could be modulated (≈2-fold increase) by elevated cellular cholesterol in normal cells. In conclusion, FHD is characterized by reduced HDL₃- and apoA-I–mediated cellular cholesterol efflux. It is not associated with abnormal cellular HDL₃ binding or a defect in a putative HDL binding protein.