ABSTRACT.    Many flagella are composed only of a microtubular structure, the axoneme, and its surrounding membrane. However supplementary (or extra-axonemal) structures can be found in many instances, in particular in protists or sperm cells. Little is known about the molecular composition and the function of these structures within the flagellum. Trypanosomes are flagellated eukaryotes, responsible for several tropical diseases (sleeping sickness, Chagas disease). In addition to the classical axoneme, their flagellum contains an extra-axonemal structure, called the paraflagellar rod (PFR), which runs alongside the axoneme. In trypanosomes it was shown that the PFR is essential for cell motility (Bastin et al, 1998, Nature 398, 548). Here we report the involvement of a kinesin belonging to the kinesin-9 family (TbKIF9) in the assembly of the PFR. Functional analysis revealed that this protein is essential for cell motility. Detailed analysis of the mutant cell line by electron microscopy shows a striking phenotype: although the axoneme is properly assembled, the PFR structure is irregular: accumulation of detergent-resistant material surrounding the axoneme in some regions whereas other regions of the axoneme are "naked", i.e. without a PFR. This is the first report of a protein specifically involved in the assembly of an extra-axonemal structure.