Rab proteins are small GTPases that control the direction and timing of vesicle fusion during intracellular trafficking between membraneous compartments. Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracellular transport pathways, is intermediate between Saccharomyces cerevisiae and mammals. TbRAB31 is a constitutively expressed T. brucei Rab protein (formerly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K, Croft SL, Field MC. TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells. J Cell Sci 1999; 112:147-156), involved in ER to Golgi transport. TbRAB31 has high homology to members of the Sec4/Ypt1 subfamily of Rab proteins from S. cerevisiae and to Rab13 and Rab11 from higher eukaryotes. Recombinant TbRAB31 binds GTP but, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein. Antibodies against TbRAB31 recognise a discrete structure located between the kinetoplast and nucleus in interphase procyclic cells; by contrast the structure is morphologically more complex in bloodstream form (BSF) parasites, consisting of at least two foci. TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always localised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules. Additional evidence suggests that TbRAB31 localises to the trypanosome Golgi complex. Firstly, the interphase position of TbRAB31 is consistent with a Golgi location. Secondly, the TbRAB31 structure is also recognised by cross-reacting antibodies to mammalian beta-coatomer protein (beta-COP), which localises to the Golgi in mammalian cells. Thirdly, the fluorescent ceramide analogue, BODIPY-TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31. The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzidine and electron microscopy.