2, 5-Dimethyl-4-hydroxy-3 (2H)-furanone (DMHF) is an important aroma compound found in many fruits such as strawberries and pineapples and it is also produced by the soy-sauce-fermenting yeast Zygosaccharomyces rouxii after the addition of D-fructose-1,6-diphosphate to yeast-peptone-dextrose nutrient media. Dilute DMHF solutions exhibit a strawberry-like flavor while DMHF concentrates have a caramel-like aroma. In media containing D-fructose-1,6-diphosphate as the sole carbon source, growth of Z. rouxii and formation of DMHF were not observed. Although Z. rouxii cells grew in media with D-glucose as the sole carbon source, DMHF was only produced when media were supplemented with D-fructose-1,6-diphosphate. The DMHF concentration always correlated with the yeast cell count and D-fructose-1,6-diphosphate concentration. Addition of CaCl2 (up to 50 g . l(-1) led to a higher DMHF concentration. Addition of Na2SO3 reduced the growth of Z. rouxii and inhibited DMHF formation. The amount of DMHF formed by 2. rouxii was not significantly affected by the addition of KH2PO4. DMHF concentrations of 5 and 10 g . l(-1) partially and completely inhibited the growth of Z. rouxii cells, respectively. Only the singly labeled furanone was formed after the addition of 1-C-13-D-fructose-1,6-diphosphate to the medium. However, unlabeled DMHF was formed in the presence of C-13(6)-D-glucose. Therefore, the carbons of the furanone originate exclusively from exogenously supplied D-fructose-1,6-diphosphate as no exchange with the internal pool of D-fructose-1,6-diphosphate occurs. This implies that DMHF is a secondary metabolite of Z. rouxii formed from D-fructose-1,6-diphosphate. We assume that at least the first step of the metabolism of D-fructose-1,6-diphosphate takes place in the cell wall or membrane of the yeast.