Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments -gluconolactone to ethanol and carbon dioxide. We propose that a novel pathway for -gluconolactone fermentation operates in this yeast. In this pathway, -gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 220.127.116.11). After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction. The remaining half of the glucose is dissimilated via glycolysis. Involvement of this novel pathway in -gluconolactone fermentation in S. bulderi is supported by several experimental observations. (i) Fermentation of -gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as -gluconolactone. Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase. (ii) High activities of an NADP -dependent glucose dehydrogenase were detected in cell extracts of anaerobic, -gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells. Gluconate kinase activity in cell extracts was negligible. (iii) During anaerobic growth on -gluconolactone, CO2 production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO2. (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in -gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in -gluconolactone metabolism.