The Smith-Lemli-Opitz syndrome (SLOS; also known as "RSH syndrome" [MIM 270400]) is an autosomal recessive multiple malformation syndrome due to a defect in cholesterol biosynthesis. Children with SLOS have elevated serum 7-dehydrocholesterol (7-DHC) levels and typically have low serum cholesterol levels. On the basis of this biochemical abnormality, it has been proposed that mutations in the human sterol Delta7-reductase (7-DHC reductase; E.C.184.108.40.206) gene cause SLOS. However, one could also propose a defect in a gene that encodes a protein necessary for either the expression or normal function of sterol Delta7-reductase. We cloned cDNA encoding a human sterol Delta7-reductase (DHCR7) on the basis of its homology with the sterol Delta7-reductase from Arabidopsis thaliana, and we confirmed the enzymatic function of the human gene product by expression in SLOS fibroblasts. SLOS fibroblasts transfected with human sterol Delta7-reductase cDNA showed a significant reduction in 7-DHC levels, compared with those in SLOS fibroblasts transfected with the vector alone. Using radiation-hybrid mapping, we show that the DHCR7 gene is encoded at chromosome 11q12-13. To establish that defects in this gene cause SLOS, we sequenced cDNA clones from SLOS patients. In three unrelated patients we have identified four different mutant alleles. Our results demonstrate both that the cDNA that we have identified encodes the human sterol Delta7-reductase and that mutations in DHCR7 are responsible for at least some cases of SLOS.