The application of the tools of molecular biology has led to a profound increase in our current understanding of the nature of the disease states associated with defects in the phenylalanine hydroxylase (PAH) gene. Over the past decade, the PAH cDNA has been cloned and the primary structure of the PAH protein has been determined. The PAH cDNA clone has served as an invaluable probe to define the molecular structure and chromosomal location of the PAH locus in both man and other organisms. Southern analysis using the PAH cDNA as a hybridization probe has revealed the presence of numerous restriction fragment-length polymorphisms (RFLPs) in the PAH gene, which have permitted the classification of normal and mutant PAH chromosomes. RFLP analysis has also permitted the implementation of prenatal diagnosis of phenylketonuria (PKU) and other related hyperphenylalaninemic disorders. Through the use of molecular cloning and polymerase chain reaction methodologies, many molecular lesions have now been identified in the PAH gene, and their association with different PAH haplotypes and disease phenotypes can now be addressed in a rational manner. Finally, the characterization of PAH mutations has enabled the population dynamics of phenylketonuria to be examined in several different populations.