Ade-H and ade-I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade-H did not have any detectable adenylosuccinate synthetase activity and ade-I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.