The proximal promoter region of the human phenylalanine hydroxylase (PAH) gene was analyzed for the presence of mutations in 122 European phenylketonuria (PKU) and hyperphenylalaninemia patients having altogether 187 uncharacterized mutant PAH alleles. This promoter fragment, which contained the most 5' transcription start site and about 300 bp upstream, was sequenced directly from polymerase-chain-reaction-amplified genomic DNA. No disease-causing mutations but three neutral nucleotide substitutions were found. A -195 T-to-C transition was present on 1% of 441 normal and 0.3% of 653 mutant chromosomes. All chromosomes that carried this transition and to which a restriction fragment length polymorphism (RFLP) haplotype had been assigned were of haplotype 1. A -71 G-to-A change and a +7 C-to-T change were always observed together and were found on 1% of 425 normal and 4% of 681 mutant chromosomes. In addition, these two transitions were found in seven heterozygote samples where the phase could not be established due to incomplete family samples. In individuals where RFLP haplotypes were known and phase could be established, these linked substitutions were associated with RFLP haplotype 9. The relatively high frequency (10-20%) of these two polymorphisms on PKU chromosomes from Great Britain, Ireland and France may reflect a relative concentration of haplotype 9 alleles among PKU chromosomes from these countries compared to the rest of Europe. The absence of disease-causing mutations within a region of the PAH gene that possesses basal promoter activity suggests that transcriptional mutations are not likely causes of PKU in Caucasian populations.