Adenine-requiring Chinese hamster ovary (CHO-K1) auxotrophs of the complementation group Ade-I were hybridized with various human cells, and hybrids were isolated under selective conditions in which retention of the complementing gene on the human chromosome is necessary for survival. Ade-I cells are deficient in adenylosuccinase activity. This enzyme carries out two independent, but similar, steps of purine biosynthesis: the removal of a fumarate from succinylaminoimidazole carboxamide ribotide to produce aminoimidazole carboxamide ribotide and the removal of fumarate from adenylosuccinate to produce AMP. These are the 9th and 13th steps of adenylate biosynthesis, respectively. Analysis of hybrids by cytogenetics and by Southern blot techniques using chromosome 22-specific DNA probes, one of which encodes an antigen expressed in human fetal brain, indicated that human chromosome 22 was 100% concordant for growth without adenine. One hybrid subclone, isolated after two successive rounds of subcloning, was found to be capable of growth without adenine; the only human chromosome present was 22. In addition, segregants that had lost the ability to grow in adenine-free media had also lost human chromosome 22. These results suggest that the human gene for adenylosuccinase resides on chromosome 22.