The African trypanosome possesses a total of 16 small GTPases of the Rab family, which are involved in control of various membrane transport events. Recently the roles of these proteins in the endocytosis and recycling of the major surface antigen of the bloodstream form, the variant surface glycoprotein (VSG), have been described but little has been reported on the roles of Rab proteins in exocytic pathways in trypanosomatids. Whilst phylogenetic analysis based on sequence similarity indicates a comparatively well conserved core set of Rab proteins, the evolutionary distance of the trypanosome lineage from crown eukaryote model systems requires direct experimental evidence to support these sequence data. By database searching we identified two further Rab genes, TbRAB1 and TbRAB2, which are the trypanosome sequence orthologues of mammalian Rab1 and Rab2, important mediators of ER to Golgi and intra-Golgi transport processes. A remarkably high level of sequence conservation is retained between the trypanosome and higher eukaryote orthologues. By immunolocalisation we find that both TbRAB1 and TbRAB2 reside on membranes in intimate association with the Golgi complex. By heterologous expression in mammalian cells we also demonstrate conservation of targeting information in the TbRAB1 and TbRAB2 proteins, whilst TbRAB1, but not TbRAB2, can complement a Ypt1(ts) conditional mutant in Saccharomyces cerevisiae. The roles of TbRAB1 and TbRAB2 in exocytosis were examined using RNAi. Suppression of TbRAB1 or TbRAB2 was strongly inhibitory to growth and most importantly both TbRAB1 and TbRAB2 were required for normal progression of VSG through the early secretory pathway. These data indicate conservation of function for these proteins between trypanosomes and crown eukaryotes.