The Smith-Lemli-Opitz Syndrome (SLOS, McKusick #270400) is an autosomal recessive multiple malformation syndrome characterized by dysmorphic facial features, mental and growth retardation, limb anomalies, and variable internal structural anomalies. Children afflicted with this disorder have a defect in cholesterol biosynthesis. Specifically, they have a defect in the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. Fibroblasts can obtain cholesterol by either de novo synthesis or from the extracellular environment by the binding and uptake of LDL. The effect of elevated levels of 7-DHC on intracellular LDL metabolism is not known. We postulated that the abnormal sterol, 7-DHC, could perturb this processes. To investigate intracellular cholesterol metabolism in SLOS fibroblasts, we analyzed filipin staining. Filipin is a fluorescent compound that binds to unesterified cholesterol. We found that, after four days of culture in lipoprotein deficient serum (LPDS), filipin staining was more intense in SLOS fibroblasts compared to controls. If filipin staining was due to an accumulation of 7-DHC, then it would be expected to increase with increased time in culture. However, filipin staining decreased to control levels after 7 days of culture in LPDS. This suggested that the increased filipin staining was due to an accumulation of LDL cholesterol rather than due to an accumulation of 7-DHC. Consistent with this observation, we found that LDL degradation by SLOS fibroblasts was decreased compared to control and heterozygous fibroblast cell lines. Furthermore, we have found that SLOS fibroblasts have lysosomal inclusion bodies similar in appearance to those found in Niemann-Pick type C. Based on these results, we propose that in addition to their primary defect in cholesterol biosynthesis, children with SLOS may have a secondary defect in intercellular LDL cholesterol metabolism.