Schizosaccharomyces pombe has two genes each encoding an isozyme of NADH-dependent glycerol-3-phosphate dehydrogenases (gpd1+ and gpd2+). To gain an insight into the function of these genes, here we constructed a gpd2 deletion mutant, in addition to the previously constructed gpd1 deletion mutant. We showed that the gpd1+ and gpd2+ gene-products are both functional in terms of the de novo glycerol synthesis. Furthermore, the gpd1(+)-mediated glycerol production is primarily responsible for the osmoregulation, but the gpd2+ gene is not. Interestingly, however, the gpd2 deletion mutant had histidine- or lysine-auxotrophy for growth on a minimal medium.