Trypanosoma brucei is a unicellular parasite that is transmitted from one mammalian host to the next by tsetse flies. The expression of many trypanosome genes is regulated during the life cycle but there is no evidence for developmental control of transcription by RNA polymerase II. T. brucei expresses at least two hexose transporter mRNAs that are developmentally regulated; we show here that specific portions of the 3'-untranslated regions are responsible for the differential expression. Different trypanosome 3'-untranslated regions, from surface protein, phosphoglycerate kinase and aldolase genes as well as the hexose transporter genes, conferred a spectrum of levels of reporter gene expression, and these activities differed between bloodstream forms and the procyclic forms that replicate in the tsetse vector. Experiments with permanently transformed cell lines showed that regulation occurs at the mRNA level. The results suggest that post-transcriptional control of mRNAs in trypanosomatids operates at several levels, and that it will not always be possible to attribute all the regulation to short RNA motifs.