Vitelliform macular dystrophy (Best's disease) is an autosomal dominant, early-onset form of macular degeneration in which the primary defect is thought to occur at the level of the retinal pigment epithelium. Genetic linkage has mapped the disease locus to chromosome 11q12-q13.1 within a 980 kb interval flanked by markers at loci D11S4076 and uteroglobin. To identify the disease gene, we systematically characterized genes from within the critical region and analysed the coding regions for mutations in 12 patients from large multigeneration Best's disease families. Following this approach, we identified a novel gene of unknown function carrying heterozygous mutations in all 12 probands. Of these, 10 result in distinct missense mutations of amino acids that are highly conserved throughout evolution, spanning a phylogenetic distance from Caenorhabditis elegans to human, and include V9M, A10T, W24C, R25Q, R218Q, Y227N, Y227C, V235M, P297A and F305S. One deletion mutation, DeltaI295, was found in two families and segregates with the disease in both cases. Northern blot analysis reveals tissue-specific expression for this gene, exclusively in the retinal pigment epithelium. In conclusion, our data provide strong evidence that mutations in the gene that we have identified cause Best's disease.