Mutations in the uroporphyrinogen III synthase (URO-synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis. Molecular analysis of the URO-synthase gene in seven unrelated CEP patients revealed eight novel mutations. These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and -86C-->A. When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild-type activity), which was thermolabile. In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping. The identification and expression of these mutations provided genotype-phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.