Vital fluorescence staining has been used in conjunction with time-lapse video image intensification microscopy to analyze the distribution and movement of endosomes, lysosomes, and mitochondria in cultured rat ovarian granulosa cells. Exposure of 5-d granulosa cell cultures to pyrene-concanavalin A (P-Con A) or 3,3'-dioctadecylindocarbocyanine-labeled low-density lipoprotein (dil-LDL) at 4°C results in the formation of randomly distributed endosomes 10 min after warming to 37°C that exhibit saltatory motion for 20 min. If granulosa cells are labeled at 4°C with both P-Con A and dil-LDL and warmed to 37°C, both ligands are found within the same endosomes which migrate centripetally to the cell center where label accumulates within phase-dense structures by 60 min. The initial endosome saltations occur over short distances (mean distance = 4.6 μm) with a mean velocity of 0.03 μm/s. Endosome saltations then cease and are followed by a gradual centripetal migration of endosomes to the cell center where they accumulate and fuse with phase-dense structures. The second phase of movement involves a continuous, unidirectional migration of endosomes over distances ranging from 5 to 40 μm at a mean velocity of 0.05 μm/s. Lysosomes were simultaneously visualized as acridine orange-staining, phase-dense structures in control cells and cells exposed to fluorescent ligands. In untreated cells, lysosomes are dispersed throughout the cytoplasm and undergo bidirectional saltations covering a mean distance of 8.7 μm with a mean velocity of 0.3 μm/s. Lysosomes redistribute centripetally to the perinuclear region of the cell by saltatory movement within 20 min of exposure to ligand. Mitochondria were visualized with the fluorescent dye rhodamine 123 in granulosa cells labeled with P-Con A and were found to redistribute to the cell center coincident with endosomes. The microtubule-disrupting agent nocodazole was found to inhibit lysosome saltations and all phases of endosome movement. Taxol, a microtubule-stabilizing agent, partially impaired lysosome movement and led to a redistribution of lysosomes into linear aggregates surrounding the nucleus. Taxol was also found to inhibit endosome movement. The data indicate that (a) endosome movement proceeds initially by saltation and later by a nonsaltatory centripetal migration in association with mitochondria, that (b) lysosomes and endosomes undergo a temporally distinct but spatially similar change in cytoplasmic distribution, and that (c) microtubules are required for the directed translocation of endosomes and lysosomes towards the cell center.