Myosin II is required for normal amoeboid locomotion. In order to understand how myosin II elicits its effects on locomotive behavior, we have mapped myosin II-cytoskeleton interactions in locomoting endothelial cells. Bovine microcapillary endothelial cells were microinjected with fluorescently labeled myosin II, and the distribution of myosin II was imaged in the living cells by fluorescence microscopy. The same cells were then permeabilized with Triton X-100 and imaged again. The second set of images showed only myosin II that was associated with detergent-insoluble cytoskeleton. Dividing the image of retained myosin II by that of total myosin II produced a map of the extent to which myosin II was associated with the detergent-resistant cytoskeleton at any point in the cell. In cells migrating at the edge of a scrape wound, myosin II was preferentially retained in a region approximately 10 microm wide located just behind the cells' leading lamellipodia. Relatively little myosin II was retained in perinuclear cytoplasm. A vector representation of the distribution of total versus retained myosin II demonstrated that myosin II retention was sharply polarized with respect to locomotion, favoring the front of migrating cells. Myosin II-enriched cytoskeleton in this region may help polarize protrusive activity and/or move cytoplasmic bulk forward. Patches of myosin II retention were also observed in adherent tails of many cells, consistent with a role in pulling the rear of the cell forward.