The fungal phylum zygomycota uses trisporic acids (TSAs), a family of apocarotenoids, during sexual reproduction to synchronize and control activity between the mycelial hyphae of opposite mating types. Separate as well as mixed cultures of Blakeslea trispora were systematically supplemented with putative, deuterium-labeled precursors downstream of beta-carotene en route to the bioactive TSAs. Analysis of the isolated metabolites allowed the reconstruction of the complete biosynthetic sequence between the first apocarotenoid, D'orenone (1), and the different series of TSAs B (8) and C (13). Both mating types produced a similar spectrum of early metabolites upstream of trisporols B (7) and C (12), while only the (+) type was able to further oxidize trisporols B (7) and C (12) to the corresponding methyltrisporoid B (5) and C (11), respectively. A novel 4-dihydrotrisporic acid B (14) that was not formed from the labeled precursors was isolated from mated strains; this compound might be derived from oxygenated beta-carotene by a parallel pathway. The ester accumulated in the culture broth of the (+) strain and was only hydrolyzed by mycelia of the (-) strain; this corresponds to a synchronization of the biosynthetic activities of both mating types.