We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dictyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5 +/- 4.9 microns/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0 +/- 4.8 microns/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5 +/- 2.0 microns/min, while wild-type cells under these conditions move at 5.9 +/- 4.5 microns/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular development. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell morphology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a multicellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.