Rab GTPases are key regulators of vesicular traffic in eukaryotic cells. Here we sought a global characterization and description of the Plasmodium falciparum family of Rab GTPases. We used a combination of bioinformatic analyses, experimental testing of predictions, structure modelling and phylogenetics. These analyses led to the identification of seven new parasite Rabs. Accordingly we estimate that the P. falciparum family is made up of 11 genes. We show that ten members of this family are transcribed in infected erythrocytes. Concerning the various members of the family, a series of specific as well as global conclusions can be drawn. Rabs predicted to be compartment-specific show different subcellular distributions. This is demonstrated for PfRab1A and PfRab11A, with the generation of specific antisera. The sequence analyses reveal several peculiarities, with possible functional implications. One of the transcribed genes, Pfrab5b, does not encode a classical C-terminus, suggestive of a novel regulatory role for this GTPase. Another, Pfrab5a, previously identified as a rab gene located on chromosome 2, possesses a 30-amino-acid insertion in its GTP-binding domain. Structural considerations suggest that this insertion could represent a novel interaction interface. We used conserved RabF and RabSF motifs to discriminate between specific parasite Rabs, and followed their predicted change in position on the structure of PfRab6, as GTP is hydrolysed to GDP. This allowed us to propose their involvement in potential interaction surfaces, that we extended to human Rab6 and the motifs known to mediate Rabkinesine-6 binding. Finally, we compared the P. falciparum Rab family to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe and found that parasite Rabs segregate into possible functional clads. Such grouping into clads may give clues to parasite Rab function, and may shed light on P. falciparum secretory/endocytic pathways. © 2003 Elsevier Science B.V. All rights reserved.