The present report gives an insight into the specific changes in the three main carotenoids (lycopene, γ-carotene and β-carotene) occurring in Blakeslea trispora cultures, with regard to medium composition. Various carbon sources and refined natural vegetable oils as co-substrates were used. The different carbon sources greatly affect the final composition of carotenoids, with lactose medium to preferentially accumulate the all-trans-β-carotene. Furthermore, the use of lactose and starch as carbon sources, respectively, gives a first indication that B. trispora is able to metabolise various homo- and hetero-saccharides, thus leading to different carotenoid percentages. The presence of oils as co-substrates resulted in enhanced fungus growth and subsequent higher carotenoid production. Substrates containing linoleic acid or other oils rich in it led to less lycopene accumulation. The data showed that the biosynthesis of lycopene, γ-carotene and β-carotene starts in most cases simultaneously in the early growth phase even in trace amounts and thus may play a role also as antioxidants for the B. trispora cells.