Familial deficiency of lecithin:cholesterol acyltransferase (LCAT) is an autosomal recessive disorder characterised by abnormalities of all plasma lipoprotein classes and by abnormal deposition of unesterified cholesterol in tissues. To elucidate the molecular basis of the disease, the LCAT genes of three unrelated Japanese patients were amplified by means of the polymerase chain reaction. Direct sequencing of the amplified fragments covering all exons and junctions showed that the patients are homozygotes for separate gene mutations. In one patient a 3 bp insertion, which should cause a substantial change in the enzyme structure, was found in exon 4; he had near absence of LCAT mass and activity. Two separate missense mutations were identified in exon 6 of the other two patients, who produced functionally defective enzymes that differed widely in specific activity. The replacement of asparagine228 with positively charged lysine completely abolished enzyme activity, whereas the other, conservative, aminoacid substitution (methionine293----isoleucine) gave rise to a partially defective enzyme. These results show that distinct mutations cause differences in plasma LCAT activity and LCAT mass, ultimately leading to differential phenotypic expression of familial LCAT deficiency.