The incorporation of radiolabeled lipid precursors into triacylglycerol (TG) molecular species in Mortierella ramanniana var. angulispora, an oleaginous fungus, was studied to determine the biosynthetic pathways for TG molecular species. Radiolabeled TG molecular species were separated and quantified by reverse-phase high performance liquid chromatography with a radioisotope detector. The major TG molecular species labeled by [1-¹⁴C] oleic acid at 30°C were OOP, OOO, and OPP (TG molecular species designations represent three constituent acyl groups. G, γ-linolenic acid; L, linoleic acid; O, oleic acid; S, stearic acid; P, palmitic acid), which were abundant TG molecular species in this fungus. The incorporation of [1-¹⁴C] oleic acid at 15°C into these molecular species was the same, while that into most other species was decreased, suggesting that biosynthesis of major molecular species such as OOP, OOO, and OPP differs from that of other TG molecular species. [1-¹⁴C] Linoleic acid incorporation indicated that the major labeled molecular species were LOP and LOO, which may be due to acylation of oleoyl, palmitoyl-glycerol, or dioleoyl-glycerol by exogenous linoleic acid. This is basically the same mechanism as for OOP and OOO biosynthesis from exogenous oleic acid. [¹⁴C(U)] Glycerol incorporation suggested that TG molecular species containing palmitic acid such as OPP were more readily synthesized through the de novo pathway. Further experiments involving inhibitors such as sodium azide and cerulenin suggested that OOO biosynthesis included a mechanism differing from that in the cases of OOP and OPP. Trifluoperazine, which inhibits the conversion from phosphatidic acid to TG, decreased [1-¹⁴C] oleic acid incorporation into all molecular species, suggesting that the incorporation into all molecular species included the de novo TG biosynthetic pathway via phosphatidic acid. These results revealed that the biosynthetic pathways for TG molecular species can be classified into several groups, which exhibit different sensitivities to low temperature and inhibitors of lipid metabolism. This implies that the composition of TG molecular species is regulated through different biosynthetic pathways responsible for specific TG molecular species, providing a new insight into the biosynthesis of TG molecular species.