Genes SNO1 and SNZ1 are Saccharomyces cerevisiae homologues of PDX2 and PDX1 which participate in pyridoxine synthesis in the fungus Cercospora nicotianae. In order to clarify their function, the two genes SNO1 and SNZ1 were expressed in Escherichia coli either individually or simultaneously and with or without a His-tag. When expressed simultaneously, the two protein products formed a complex and showed glutaminase activity. When purified to homogeneity, the complex exhibited a specific activity of 480 nmol.mg(-1).min(-1) as glutaminase, with a Km of 3.4 mm for glutamine. These values are comparable to those for other glutamine amidotransferases. In addition, the glutaminase activity was impaired by 6-diazo-5-oxo-L-norleucine in a time- and dose-dependent manner and the enzyme was protected from deactivation by glutamine. These data suggest strongly that the complex of Sno1p and Snz1p is a glutamine amidotransferase with the former serving as the glutaminase, although the activity was barely detectable with Sno1p alone. The function of Snz1p and the amido acceptor for ammonia remain to be identified.