The effect of inoculum, pH, carbon and nitrogen source, natural oils, fatty acids, antioxidant, and precursors on beta-carotene production by Blakeslea trispora in shake-flask culture was investigated. The highest concentration of beta-carotene was obtained in the medium (pH 7.0) inoculated with one loop of each culture. Sucrose, glycerol, cornmeal, soy protein acid hydrolysate, and distiller's solubles did not improve the production of beta-carotene. By contrast, glucose, corn steep liquor, antioxidant, olive oil, soybean oil, cottonseed oil, oleic and linoleic acids, and kerosene significantly increased the gamma-carotene production. A central composite design was employed to determine the maximum P-carotene production at optimum values for the process variables (linoleic acid, kerosene, and antioxidant). The fit of the model was found to be good. Linoleic acid, kerosene, and antioxidant had a strong linear effect on beta-carotene production. The concentration of beta-carotene was significantly affected by linoleic acid-kerosene and linoleic acid-antioxidant interactions as well as by the negative quadratic effects of these variables. The interaction between kerosene and antioxidant had no significant linear effect. The maximum beta-carotene concentration (2.88 g/L) was obtained at concentrations of 17.15 g/L of linoleic acid, 39.25 g/L of kerosene, and 9.04 g/L of antioxidant.