Our studies on the in vitro differentiation of a pleomorphic Trypanosoma brucei strain TREU667 indicate that the parasite differentiates directly from long-slender into procyclic form when incubated in Cunningham's medium at 26 degrees C. The intermediary and the short-stumpy bloodstream forms harvested from infected mice can also differentiate directly into procylic form in vitro with time courses similar to that for the long-slender form. Thus, none of the three bloodstream forms appear to be significantly better preadapted for differentiation. Tricarboxylic acid (TCA) cycle intermediates cis-aconitate and L-citrate can shorten the initial lag phase of the differentiation, but an essential trigger appears to be the temperature shift from 37 degrees C to 26 degrees C, when other TCA cycle intermediates such as L-proline, L-malate, alpha-ketoglutarate, fumarate and succinate are present in Cunningham's medium. The ornithine decarboxylase (ODC) activity in T. brucei showed a gradual increase and the ODC mRNA level remained constant during the differentiation. DL-alpha-Difluoromethylornithine (DFMO), putrescine, dibutyryl cAMP and theophylline all exerted no discernible effect on the in vitro process, which suggests that neither cAMP increase nor polyamine depletion could be counted among the triggers of T. brucei differentiation. A monomorphic T. brucei strain EATRO110 was tested in the same medium at 26 degrees C but was unable to differentiate.