Small G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport. Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material. Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis. Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified. Here we report cloning of a second Rab5 homologue, TbRab5Bp. The TbRAB5A and -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently. Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form. mRNA levels of TbRab5A and -4 are constitutive. Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF. Also, the TbRab5B mRNA has a large 3'-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites. Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket. TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG100), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway. TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s. Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence.