Optimization of the medium components which enhance carotene production by Blakeslea trispora was achieved with the aid of response surface methodology. In the first step, a central composite design was employed to achieve the highest carotene concentration at optimum values of the process variables, i. e., linoleic acid, Span 20, and butylated hydroxytoluene (BHT), added in the basal medium. The fit of the model was found to be significant. The production medium to achieve the highest carotene concentration (139. 0 ± 4. 5 mg/g dry biomass) was composed of the basal medium supplemented with linoleic acid (21. 3 g/l), Span 20 (16. 0 g/l), and BHT (4. 7 g/l). The results show that the optimization strategy led to an increase in carotene production by 35-fold. The carotenes content in Β. trispora were β-carotene (45%), γ-carotene (31%), and lycopene (24%). In the second optimization step, the production medium was supplemented with different trace elements which significantly affect carotene production (i. e., CuSO ₄ ^.5H ₂O, FeCl ₃ ^.6H ₂O, and Co(NO ₃) ₂.6H ₂O). The experimental validation showed that the model was effective. The optimized medium for enhanced carotene concentration consists of the production medium supplemented with 395. 64 mg/l CuSO ₄ ^.5H ₂O, 10. 0 mg/l FeCl ₃ ^.6H ₂O, and 1. 12 mg/l Co(NO ₃) ₂ ^.6H ₂O. Practical validation of the above optimum medium gave carotene production 154. 0 ± 5. 0 mg/g dry biomass, which is 10% higher than the concentration of carotenes in the production medium. In this case, the carotenes consisted of β-carotene (37%), γ-carotene (47%), and lycopene (16%). Thus, the addition of trace elements to the production medium increased slightly the concentration of carotenes, but changed mainly the composition of the carotenes to a drastic increase of γ-carotene concentration.