Primary congenital glaucoma (GLC3) is an inherited eye disorder that accounts for 0.01-0.04% of total blindness. Although a large number of chromosomal abnormalities have already been reported in patients with congenital glaucoma, the precise location and pathogenesis of this condition remain elusive. By using a group of 17 GLC3 families and a combination of both candidate regional and general positional mapping strategies, we have mapped a locus for GLC3 to the short arm of chromosome 2. Eleven families showed no recombination with 3 tightly linked markers of D2S177 (Z = 9.40), D2S1346 (Z = 8.83), and D2S1348 (Z = 8.90) with a combined haplotype lod score of 11.50. Haplotype and multipoint linkage analyses of 14 DNA markers from 2p indicated that the disease gene is located in the 2p21 region and is flanked by DNA markers D2S1788/D2S1325 (theta = 0.03; Z = 5.42) and D2S1356 (theta = 0.05; Z = 4.69). Inspection of haplotype and heterogeneity analysis confirmed that 6 families are not linked to the 2p21 region, thus providing the first proof of genetic heterogeneity for this phenotype. We therefore designated the locus on 2p21 GLC3A and positioned it in the overall linkage map of Tel-D2S405-D2S367-(D2S1788/D2S1325)-[(GLC3A, D2S177)/(D2S1346/D2S1348)]-D2S1356-D2S119-D2S1761-D2S1248-D2S1352-D2S406-D2S441-Cen. Of the seven genes mapping to the 2p21 region, CAD, CALM2, and LHCGR are centromeric to D2S119 and can be excluded as a candidate for GLC3A, but mutations in PRKR, TIK, SOS1, or SPTBN1 may still be accountable for this phenotype. As human 2p21 shows homology with mouse chromosomes 11 and 17, the homolog of GLC3A is expected to reside on one of these two chromosomes. (C) 1995 Academic Press, Inc.