Chinese hamster ovary cells were treated with various physical and chemical mutagens and subject to the bromodeoxyuridine plus visible light procedure for isolation of auxotrophic mutants. more than 200 auxotrophs have been isolated which require exogenous supplement of various nutrilites for growth, such as glycine, adenine, thymidine, inositol, etc. Fifty-five of these have been characterized by complementation tests and were shown to constitute 15 different loci. All these auxotrophs are highly stable and exhibit all-or-none growth response to the respective nutrilites. Enzyme deficiencies have been identified in several of these classes. When these auxotrophs are hybridized with human cells and grown in selective medium, the hybrids lose human chromosomes at a rapid rate and the analysis for synteny of the human genes can be successfully carried out. Moverover, in hybrids formed between an adenine-requiring auxotroph and human cells, a human esterase activator gene has been identified which appears to regulate the expression of esterase gene activities in the Chinese hamster genome. Studies of this kind may lead to the understanding of gene regulation in mammalian cells.