The elucidation of the molecular basis of hyperphenylalaninemia in various world populations (PKU Consortium Database: http://www.mcgill/ca/pahdb/) has revealed a remarkable molecular heterogeneity at the locus encoding for phenylalanine hydroxylase. As a consequence, genotyping of HPA patients has prompted the establishment of an impressive number of mutatIon detection protocols. In spite of the large variety of methods proposed so far, no comprehensive strategy has been yet developed for the detection of PAH gene mutations. Therefore, new approaches, combining the advantages of individual methods are required, especially in populations with a high number of PAH gene mutations. In this study, we propose the use of Reverse Dot Blot Analysis within a general mutation protocol to simplify the genotyping of hyperphenylalaninemics in the very heterogeneous population of Sicily (Italy).