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Karyological analysis was performed on a series of human-Chinese hamster cell hybrids containing deletions of human chromosome 12. Chromosome breakage was produced by treatment of the cells with either X-rays or 5-bromodeoxyuridine and near-visible light. The hybrid clones were analyzed for the presence or absence of the following five human gene markers known to be located on chromosome 12: triosephosphate isomerase-1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase-B (LDHb), serine hydroxymethyltransferase (SHMT), and peptidase-B (PEPB). Based on the correlation between the isozyme markers and karyological analysis of these clones, a regional map of the five human genes on chromosome 12 was established. The linear order for these genes is: pteτ-TPI1-GAPDH-LDHB-centromere-SHMT-PEPB-qteτ. The locations of these genes are: TPI1, GAPDH, LDHB: pter→p12; SHMT: q12→q14; PEPB: ql4→qter. Statistical analysis similar to that of Goss and Harris (1975, 1911a, b) has been performed on the segregation data in the hybrid clones. The statistical map, in general, agrees with the cytogenetic map and further localizes PEPB to 12q21.