A plethora of different mutations in the gene for the low density receptor (LDLR) are responsible for the autosomal dominant inherited disorder familial hypercholesterolemia (FH). However, only a few splice site mutations have been identified in this gene. We here report a defect presumably affecting the splicing of precursor mRNA, resulting from a novel mutation, a G to A transition at the terminal nucleotide of intron 12, of the LDLR gene detected in three unrelated families with heterozygous FH. This mutation markedly reduced the steady-state transcript level of the mutant LDLR allele as compared to the corresponding normal LDLR allele in heterozygous FH patients as measured by a fluorescence based, allele-specific quantitation technique. In the FH families, the acceptor splice site mutation cosegregates with hypercholesterolemia, and it is associated with onset of ischemic heart disease in the fifth and sixth decade of life.