Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi occurs by an actin-independent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attachment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypothesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to form the parasitophorous vacuole. Here we directly demonstrate directional migration of lysosomes to the parasite entry site, using time-lapse video-enhanced microscopy of L6E9 myoblasts exposed to T. cruzi trypomastigotes. BSA-gold-loaded lysosomes moved towards the cell periphery, in the direction of the parasite attachment site, but only when their original position was less than 11-12 microns from the invasion site. Lysosomes more distant from the invasion area exhibited only the short multi-directional saltatory movements previously described for lysosomes, regardless of their proximity to the cell margins. Specific depletion of peripheral lysosomes was obtained by microinjection of NRK cells with antibodies against the cytoplasmic domain of lgp 120, a treatment that aggregated lysosomes in the perinuclear area and inhibited T. cruzi entry. The microtubule-binding drugs nocodazole, colchicine, vinblastine, and taxol also inhibited invasion, in both NRK and L6E9 cells. Furthermore, microinjection of antibodies to the heavy chain of kinesin blocked the acidification-induced, microtubule-dependent redistribution of lysosomes to the host cell periphery, and reduced trypomastigote entry. Our results therefore demonstrate that during T. cruzi invasion of host cells lysosomes are mobilized from the immediately surrounding area, and that availability of lysosomes at the cell periphery and microtubule/kinesin-mediated transport are requirements for parasite entry.