A mutant cell line, called don801, was isolated from a wild-type population of V79.5 Chinese hamster cells by its ability to grow in the presence of the glutamine analog 6-diazo-5-oxo-L-norleucine (DON), which is toxic for V79.5 cells. The don801 cells were found not to be cross-resistant to another glutamine analog, O-diazoacetyl-L-serine (azaserine, AS). It was shown that guanine but neither hypoxanthine nor adenine protected V79.5 cells from the toxic effects of DON, while hypoxanthine and adenine, but not guanine protected them against AS toxicity. Exposure of wild-type cells to DON was shown to result in a specific reduction of intracellular GTP pools, while in the mutant cells there was no effect on GTP levels. These results strongly suggested that DON was specifically inhibiting guanylate synthetase (GMP synthetase;xanthosine-5'-phosphate: L-glutamine amidoligase, EC 188.8.131.52) in V79.5 cells and that the enzyme in don801 cells was resistant to inhibition. In vitro assays of GMP synthetase activities from V79.5 and don801 cells confirmed this hypothesis. The mutant phenotype was also found to be dominant in intraspecific cell hybrids.