Somatic hybrids between human cells and the Chinese hamster ovary (CHO) K1 mutant, Ade -H cells, were selected for purine prototrophy by growth in adenine-free medium. The Ade -H mutant is defective in the enzyme adenylosuccinate (AMPS) synthetase (ADSS; EC 6.3.4.4), which carries out the first of a two-step sequence in the biosynthesis of AMP from IMP, and therefore requires exogenous adenine for growth. The presence of the long arm of human chromosome 1 in the hybrids is 100% concordant for the ability to grow in adenine-free medium and restoration of the enzyme activity. Hybrid segregants that lose the ability to grow in adenine-free medium lose all or a portion of chromosome 1 and enzyme activity. Southern blot hybridization with a chromosome 1-specific probe, BCMI, confirms the existence of human chromosome 1 in these hybrids. Analysis of a human/CHO translocation chromosome that arose in one of the hybrids suggests that the gene correcting the defect lies in the region 1 cen-1q12. In summary, we have shown by cytogenetics, segregant analysis, biochemical assay, and Southern blot analysis that human chromosome 1, most likely in the region 1cen-1q12, corrects the defect in ADSS-deficient mutant Ade-H cells.