Kinesins are cytoskeletal motor proteins which play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned and functionally characterized in Trypanosoma brucei (T.brucei) a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 µM for the microtubule dissociation constant (kd) with a Kcat of 0.2 s-1. Immunolocalisation analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH4Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo we produced an inducible kinesin deficient strain by dsRNA interference methodology. Mutant cells were loaded with the fluorescent reagent fura2-AM to measure intracellular free calcium ([Ca++]i). The resting [Ca++]i was unchanged in mutant cells, however alkalinisation of acidic vesicles induced by NH4Cl or nigericin was not followed by release of Ca++. These data and the relative importance of the ionomycin-releasable and the ionomycin-plus-NH4Cl-releasable Ca++ pools suggest a lower Ca++ content in acidocalcisomes and dysfunctional Ca++ release.