Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway, a reaction that generates NADPH. We have isolated ZWF1, the Saccharomyces cerevisiae gene that encodes G6PD, and identified its transcript and transcription start point. Expression of ZWF1 appears not to be regulated, consistent with its 'housekeeping' role. Null mutants lacking G6PD appear to grow normally, but are more sensitive than wild type to oxidizing agents that presumably reduce the level of NADPH. This suggests that G6PD has a major role in NADPH production in yeast. Regulation of GAL1 expression appears normal in zwf1 mutants, suggesting that the pentose phosphate pathway is not involved in glucose repression. The predicted amino acid sequence of yeast G6PD is highly similar to the sequence of the Drosophila, human, and rat enzymes, except near its N terminus, where the yeast and Drosophila sequences diverge from that of human and rat.