We present a quantitative description of the molecular species composition of the major phospholipid classes in bloodstream and procyclic forms of Tripanosoma brucei. Phospholipid classes were resolved by 2-dimensional thin-layer chromatography. Diradylglycerols were released from individual phospholipid classes by phospholipases C, converted into benzoate derivatives and separated into diacyl, alkylacyl and alk-1-enylacyl subclasses. Individual molecular species were quantitated and identified by HPLC and the assignments were confirmed by mass spectrometry. Comparison of the diacyl species of PC, PE and PI in bloodstream trypanosomes showed major differences in the relative amounts of individual molecular species between the different classes but no striking changes in the degree of saturation or overall chain length. In contrast, in procyclic trypanosomes the relative amounts of diacyl molecular species with polyunsaturated fatty acyl chains decreased in the order of PC> PE> PI. Also, the alkylacyl and alk-1-enylacyl subclasses of PC and PE in bloodstream trypanosomes comprised a single molecular species, 18:0 18:2. Such exclusivity was not observed in procyclic trypanosomes among the same phospholipid subclasses, although 18:0 18:2 was the predominant species. Almost all the PI of bloodstream forms contained one 18:0 acyl species, which is consistent with the composition of the PI used for glycosylphosphatidylinositol synthesis. (C) 1993 Elsevier Science B.V. All rights reserved.