We have localised, within a Saccharomyces cerevisiae genomic fragment, the GUA1 gene whose amplification leads to the accumulation of several polypeptides on the two-dimensional (2-D) map of yeast proteins. Comparison of the sequence of the putative GUA1 protein with a data library shows a strong similarity with Escherichia coli, Bacillus subtilis and Dictyostelium discoideum GMP synthetases (GMPS) and other glutamine amidotransferases. The fact that disruption of the chromosomal copy of the gene leads to guanine auxotrophy, that the gual::URA3 disruption does not complement an independently obtained gual-3 mutation deficient in GMPS and that GUA1 complements this latter mutation, confirms the identification of the cloned gene as GUA1 encoding the S. cerevisiae GMPS. Finally, using microsequencing, we have identified one of the polypeptides, which is overproduced in response to GUA1 amplification, as corresponding to GUA1.