4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) production by Zygosaccharomyces rouxii was studied under various culture conditions. Among the precursors added, D-fructose 1,6-bisphosphate was the best. Aerobic conditions led to a higher HDMF level, and the concentration of the added D-fructose 1,6-bisphosphate up to 10% increased HDMF production; HDMF biosynthesis then decreased and was strongly inhibited by 20% of D-fructose 1,6-bisphosphate. HDMF was produced by Z. rouxii during the growth phase and the stationary phase. This microbiological method enabled the biosynthesis of ''natural'' HDMF at 100 ppm.