The effect of aeration rate and agitation speed on beta-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentor and optimization of the production of the pigment in a bubble column reactor were investigated. In addition, a central composite design was employed to determine the maximum beta-carotene concentration at optimum values for the process variables (aeration rate, sugar concentration, linoleic acid, kerosene). By image analysis of the morphology of the fungus, a quantitative characterization of the hyphae and zygospores formed was obtained. The hyphae were differentiated to intact hyphae, vacuolated hyphae, evacuated cells and degenerated hyphae. An increased proportion of zygospores was correlated to high beta-carotene production. In the stirred-tank fermentor, the highest concentration of the carotenoid pigment (92.0 mg/L) was obtained at an aeration rate of 1.5 vvm and agitation speed of 60 rpm. In the bubble column reactor, the aeration rate and concentration of sugars, linoleic acid, kerosene, and antioxidant significantly affected the production of beta-carotene. In all cases, the fit of the model was found to be good. Aeration rate, sugar concentration, linoleic acid, and kerosene had a strong positive linear effect on beta-carotene concentration. Moreover, the concentration of the pigment was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. Maximum beta-carotene concentration (360.2 mg/L) was obtained in culture grown in molasses solution containing 5% (w/v) sugar supplemented with linoleic acid (37.59 g/L), kerosene (39.11 g/L), and antioxidant (1.0 g/L).