AbstractThis review summarizes the isolation of rat phenylalanine hydroxylase mRNA and its use in the synthesis of its cDNA. As rat cDNA cross‐hybridized with human phenylalanine hydroxylase mRNA, the rat cDNA probe was used to screen a human liver cDNA library. A partial length cDNA human phenylalanine hydroxylase probe was obtained which showed restriction fragment length polymorphism (RFLP) with 3 restriction enzymes and was successfully used to trace the transmission of the mutant gene in PKU families with one or more affected children. Recently the partial‐length cDNA probe has been used to isolate a full‐length cDNA probe for human phenylalanine hydroxylase. Gene transfer experiments with the full‐length cDNA have led to expression of human phenylalanine hydroxylase in eukaryotic cultured cells and in recombinant bacteria which normally do not express phenylalanine hydroxylase activity. The full‐length cDNA of human phenylalanine hydroxylase has been sequenced, uncovering the nucleic acid sequence of the exons of the human phenylalanine hydroxylase gene, as well as the most likely amino acid structure of the human phenylalanine hydroxylase enzyme. The full‐length cDNA probe has 10 identifiable binding sites for restriction enzymes that show RFLP. These additional RFLPs have enabled haplotype analyses of the normal and mutant phenylalanine hydroxylase genes in PKU families. Haplotype analyses in Danish PKU families revealed 12 different haplotypes. However, of 132 chromosomes analysed from 66 obligate heterozygotes, 59 out of 66 PKU genes were associated with only 4 haplotypes. Cosmid cloning and preliminary characterization of the human phenylalanine hydroxylase gene have identified 13 exons distributed across a gene that is more than 190 kb in length. In β‐thalassaemia, distinct mutations in the β‐globin locus are associated with specific RFLP haplotypes within a given population. As in thalassaemia such an association forms a strategy for cloning and sequence characterization of mutant phenylalanine hydroxylase genes derived from each haplotype. If the PKU genes in the Danish population are the result of mutiple mutations which occurred on chromosomes of the most common haplotypes, the same strategy is potentially applicable for the molecular characterization of the various types of phenylalanine hydroxylase deficiency.