Erythropoietic protoporphyria (EPP) is characterized by excess accumulation of protoporphyrin, which is due to deficient activity of the enzyme ferrochelatase (FECH). This results in photosensitivity and in some patients liver disease which may necessitate liver transplantation. The aim of this study was to delineate the abnormalities in the FECH gene which cause phenotypic expression in EPP. We identified 43 individuals from 25 North American families with EPP who were heterozygous for various FECH mutations, but the mutations did not adequately explain the variable phenotype. We also examined the presence of an intron polymorphism (IVS3-48c) in the FECH gene which was shown to cause the formation of aberrantly spliced FECH mRNA. FECH DNA analysis demonstrated that 94% of 31 symptomatic individuals with FECH mutations were heterozygous for IVS3-48c, whereas 12 asymptomatic individuals with FECH mutations were homozygous for IVS3-48t. Haplotype analysis in four families showed that symptomatic members had the IVS3-48c polymorphism in the non-mutant FECH allele. Sequencing of the proximal FECH gene promoter showed no additional changes which might affect gene expression. The levels of normal FECH mRNA, measured by relative quantitative RT-PCR, and FECH enzyme activity were correspondingly lower in the cultured lymphoblasts of family members with the IVS3-48c polymorphism. These results indicate that symptomatic disease in most North American patients with EPP is explained by the inheritance of a mutation in one FECH allele which causes a structural alteration in the protein, together with a low expressing non-mutant FECH allele which is caused by the IVS3-48c polymorphism.