BACKGROUND: The increasing industrial demand for carotenoids has aroused interest in their bio-production, and the need to reduce production costs has encouraged the use of low cost industrial substrates, such as agro-industrial residues. Thus the objective of this research was the bio-production of carotenoids by Sporidiobolus salmonicolor using agro-industrial substrates (corn steep liquor and sugarcane molasses), pre-treated with acids (sulphuric and phosphoric). RESULTS: Bio-production was carried out in an orbital shaker using a 10% (v/v) inoculum, incubation at 25 degrees C, and agitation at 180 rpm for 120 h in a non-illuminated environment. The carotenoids were recovered using liquid N-2 combined with dimethylsulphoxide for cell rupture, and an acetone/methanol mixture (7:3 v/v) for extraction. CONCLUSION: The complete second-order design allowed for optimisation of the carotenoid concentration obtained from industrial substrates pre-treated with acids (sulphuric and phosphoric), obtaining a total carotenoid content of 541.5 mu g L-1 using 10 g L-1 sugarcane molasses, 5 g L-1 corn steep liquor and 5 g L-1 yeast hydrolysate at 25 degrees C, with agitation at 180 rpm and an initial pH of 4.0. (C) 2008 Society of Chemical Industry.