Knobloch syndrome (KS) is an autosomal recessive disorder defined by the occurrence of high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. The KS causative gene had been assigned to a 4.3 cM interval at 21q22.3 by linkage analysis of a large consanguineous Brazilian family. We reconstructed the haplotypes of this family with ten additional markers (five were novel) and narrowed the candidate interval to a region of <245 kb, which contains 24 expressed sequence tags, the KIAA0958 gene and the 5' end of the COL18A1 gene. We identified a homozygous mutation at the AG consensus acceptor splice site of COL18A1 intron 1 exclusively among the 12 KS patients, which was not found among 140 control chromosomes. This mutation predicts the creation of a stop codon in exon 4 and therefore the truncation of the alpha1(XVIII) collagen short form, which was expressed in human adult retina. These findings provide evidence that KS is caused by mutations in COL18A1 which, therefore, has a major role in determining the retinal structure as well as in the closure of the neural tube. Therefore, we show for the first time that the absence of a collagen isoform impairs embryonic cell proliferation and/or migration as a primary or secondary effect.