The classic phenotype of Fabry disease, X-linked alpha -galactosidase A (alpha -Gal A) deficiency, has an estimated incidence of approximately 1 in 50,000 males. The recent recognition of later-onset variants suggested that this treatable lysosomal disease is more frequent. To determine the disease incidence, we undertook newborn screening by assaying the alpha-Gal A activity in blood spots from 37,104 consecutive Italian male neonates. Enzyme-deficient infants were retested, and "doubly screened-positive" infants and their relatives were diagnostically confirmed by enzyme and mutation analyses. Twelve (0.03%) neonates had deficient alpha-Gal A activities and specific mutations, including four novel missense mutations (M51I, E66G, A73V, and R118C), three missense mutations (F113L, A143T, and N215S) identified previously in later-onset patients, and one splicing defect (IVS5(+1G-->T)) reported in a patient with the classic phenotype. Molecular modeling and in vitro overexpression of the missense mutations demonstrated structures and residual activities, which were rescued/enhanced by an alpha-Gal A-specific pharmacologic chaperone, consistent with mutations that cause the later-onset phenotype. Family studies revealed undiagnosed Fabry disease in affected individuals. In this population, the incidence of alpha-Gal A deficiency was 1 in approximately 3,100, with an 11 : 1 ratio of patients with the later-onset : classic phenotypes. If only known disease-causing mutations were included, the incidence would be 1 in approximately 4,600, with a 7 : 1 ratio of patients with the later-onset : classic phenotypes. These results suggest that the later-onset phenotype of Fabry disease is underdiagnosed among males with cardiac, cerebrovascular, and/or renal disease. Recognition of these patients would permit family screening and earlier therapeutic intervention. However, the higher incidence of the later-onset phenotype in patients raises ethical issues related to when screening should be performed--in the neonatal period or at early maturity, perhaps in conjunction with screening for other treatable adult-onset disorders.