We have identified a rare mutation (T-45C) in the low density lipoprotein (LDL)-receptor gene in a Welsh patient with a clinical diagnosis of heterozygous familial hypercholesterolaemia (FH). The mutation is in the proximal Sp1 binding site in repeat 3 of the 42 bp region of the promoter required for sterol-dependent regulation of transcription, but the substituted nucleotide is not a strongly conserved base in the consensus sequence for Sp1 binding. Normal and mutant promoter fragments (from base -600 to -5) were linked to a luciferase reporter gene, and transient expression in COS cells showed that the mutation reduced transcriptional activity to approximately 43% of normal in the presence, and 25% in the absence of sterols in the medium. Competitive gel-shift mobility assays showed that the mutation reduced the binding affinity for transcription factor Sp1. Analysis of a neutral polymorphism in the LDL-receptor mRNA from the patient's lymphoblasts showed that expression of one allele was reduced. Since Southern blotting of genomic DNA and sequencing of the entire coding region of the LDL-R gene did not reveal any other potential defects, we infer that the T-45 C mutation is the underlying cause of hypercholesterolaemia in the proband.