An HPLC-MSn system was used to quantify and identify the structures of individual sophorolipid components produced in Torulopsis bombicola fermentation on glucose with or without hexadecane or soybean oil. With glucose alone, the SL production was minimal and the products were complex mixtures with mainly acidic SLs. The SLs produced with glucose plus soybean oil were also complex, containing both lactonic and acidic SLs with saturated and unsaturated C16 and C18 fatty acid moieties. The glucose plus hexadecane system gave the highest production rate and product selectivity, forming primarily two diacetylated lactonic isomers with palmitate as the fatty acid moiety. A close structure correspondence between the SL's lipid moiety and the lipid precursor used was observed. The change of the composition of SL mixtures along batch fermentation was further examined. The concentrations of acidic SLs increased very gradually throughout the process. The production of lactonic SLs became appreciable following the addition of hexadecane or soybean oil at 24 h, and increased much more rapidly after the culture reached the stationary phase. The combined percentage of the main lactonic SLs leveled off at similar to 80% for the hexadecane system and similar to 50% for the soybean oil system. The yields of crude SLs were 0.84, 0.20, and 0.03 g per gram of hexadecane, soybean oil, and glucose consumed during the SL production phase. Hexadecane is thus a more efficient second C-source for sophorolipid production.