Familial hypercholesterolemia (FH) is caused by mutations in the low-density lipoprotein (LDL) receptor gene. We used a multiplex-PCR based single-strand conformation polymorphism analysis to screen the promotor region and all 18 exons of the LDL receptor gene for mutations in patients clinically diagnosed as having FH to identify their particular gene defect. An affected proband was found to be heterozygous for a missense mutation, replacing guanine to adenine at nucleotide 1171 of exon 8 of the gene. This mutation is predicted to cause a substitution of alanine to threonine at codon 370 (A370T). The base exchange abolishes a HaeIII restriction site. PCR of exon 8 followed by restriction enzyme digestion with HaeIII conveniently allowed to screen the whole family and 101 unrelated normocholesterolemic subjects from the same ethnic background for the presence of this mutation. The A307T mutation did not cosegregate with the clinical phenotype of hypercholesterolemia in the affected family. Furthermore the mutation was identified in a heterozygous state in 12 out of 101 and in a homozygous state in 1 out of 101 normocholesterolemic subjects, indicating that this mutation is a frequent genetic variation of the LDL receptor gene in the population examined. Lipid values of probands carrying the wild type LDL receptor allele and the mutation in a heterozygous state did not differ significantly. The proband carrying the mutation on both LDL receptor alleles had high normal cholesterol and LDL cholesterol levels. The A370T mutation represents the third single-amino acid change of the LDL receptor protein reported so far, which, in a heterozygous state, is not associated with the clinical phenotype of familial hypercholesterolemia. The pathophysiologic significance of homozygosity for this mutation remains to be clarified.