We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described "South African" G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.