Primary congenital glaucoma (Buphthalmos) is an autosomal recessive eye disorder, postulated to result from developmental defects in the anterior eye segment. Previously, we reported two chromosomal locations for this condition on 2p21 (GLC3A) and 1p36 (GLC3B) respectively. In this study, heritable mutations of human cytochrome P4501B1 gene (CYP1B1) in affected individuals of five well-characterized families linked to the GLC3A locus are described. CYP1B1 gene has previously been mapped within the GLC3A candidate region and its expression in the trabecular meshwork cells has been demonstrated in this study. Three different homozygous mutations were identified and characterized: a 13 bp deletion in exon III; an insertion of a single cytosine base in exon II; and a larger deletion affecting the 5' end of exon III and the adjacent intronic region. All of these are frameshift mutations that are predicted to remove domains essential for the function of the CYP1B1 protein. Therefore, it is expected that all these mutations result in functional null alleles. The mutations detected in the affected members of these families were not present in 470 chromosomes from randomly selected normal individuals, thus strongly suggesting that CYP1B1 is the gene for the GLC3A locus on 2p21. The results are discussed in the context of the earlier hypothesis that 'drug-metabolizing' enzymes might modulate the processes of growth and differentiation by controlling the steady-state-levels of oxygenated growth-effector molecules.