The carotenoid-hyperproducing mutant 2A2N of the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) was cultivated using sugar beet blackstrap molasses. This molasses was composed of 70% (w/v) total solid and 50% (w/v) total sugar. Biomass yield (biomass/carbohydrate) significantly decreased at >5% (v/v) molasses. Atomic emission spectrometry revealed that Na and P were the limiting nutrients when molasses was used. Molasses (5%, v/v) containing urea (30 g/l molasses) and sodium phosphate (NaH2PO4, 5 g/l molasses) was formulated for biomass production by the mutant. The optimal pH for carotenoid production was 4.9 during the growth phase and 2.6-3.5 during the stationary phase. The three main sugars in molasses (sucrose, glucose, and fructose) were assimilated by the mutant but fructose was consumed slowly. When the formulated medium with pH 4.5-5.5 was used, the maximal biomass yield was 36 g/l (0.18 g of yeast l(-1)h(-1) and 40 mg of carotenoid l(-1)) in fed-batch pilot-scale 100-l cultivation.