The effect of pretreatment of molasses, nitrogen sources, natural oils, fatty acids, antioxidant, precursors, and mixtures of the above substances on beta-carotene production by Blakeslea trispora in shake flask culture was investigated. Also, a central composite design was employed to determine the maximum beta-carotene concentration at optimum values for the process variables (linoleic acid, kerosene, antioxidant). The highest concentration of the carotenoid pigment was obtained in molasses solution treated with invertase. Corn steep liquor and yeast extract at concentrations of 5.0% and 0.5% (w/v), respectively, increased slightly the concentration of beta-carotene, while the natural oils, fatty acids, and precursors (except kerosene) did not improve the production of pigment when they were added separately to the medium. On the other hand, the mixture of linoleic acid, kerosene and antioxidant increased significantly the concentration of beta-carotene. The fit of the model was found to be good. Linoleic acid, kerosene and antioxidant had a strong linear effect on beta-carotene concentration. The concentration of beta-carotene was significantly affected by linoleic acid-antioxidant and kerosene-antioxidant interactions as well as by the negative quadratic effects of these variables. The interaction between linoleic acid-kerosene had no significant linear effect. Maximum P-carotene concentration (790.0 mg dm(-3)) was obtained in culture grown in molasses solution supplemented with linoleic acid (30.74 g dm(-3)), kerosene (27.79 g dm(-3)) and antioxidant (10.22 g dm(-3)).