In order to identify the molecular basis of phenylketonuria (PKU) in Italy, we screened the entire coding sequence of the phenylalanine hydroxylase gene in 20 Italian PKU patients, whose origins are scattered throughout Italy. The frequency of each identified mutation and of 5 other European mutations was determined within a panel of 92 Italian PKU patients. This approach allowed us to identify 20 different PKU mutations and characterize 64% of the Italian PKU chromosomes. Eleven mutations (IVS10nt546, L48S, R158Q, R261Q, P281L, R261X, R252W, delta T55, IVS7nt1, IVS12nt1, Y414C) represent 55.4% of the Italian PKU alleles, the most common mutations being IVS10nt546 (12.4%) and L48S (9%). All the other mutations are very rare. These data confirm the great heterogeneity expected from previous RFLP haplotype studies. Genotype/phenotype correlation allowed for assessment of the clinical impact of the 20 identified mutations.