Aims: To study the effect of intermediates of the tricarboxylic acid (TCA) cycle on the production of zeaxanthin from Flavobacterium multivorum in order to optimize production of this xanthophyll carotenoid. Methods and Results: The concentration of selected TCA cycle intermediates (malic acid, isocitric acid and alpha-ketoglutarate) was optimized in shake flask culture, using a statistical two-level, three-variable factorial approach. The carotenoid production profile was also studied in the optimized medium at various growth phases. Optimized medium resulted in a sixfold increase in volumetric production of zeaxanthin (10.65 +/- 0.63 mug ml(-1)) using malic acid (6.02 mm), isocitric acid (6.20 mm) and alpha-ketoglutarate (0.02 mm). The majority of zeaxanthin was produced in the late logarithmic growth phase whereas a substantial amount of beta-cryptoxanthin and beta-carotene were observed in the early logarithmic phase. Significance and Impact of the Study: This study demonstrates improvement of zeaxanthin production from F. multivorum which might aid in the commercialization of zeaxanthin production from this microbe.