Myosin I1 function has been implicated in non-muscle cell behaviors including movement, cytokinesis, and multicellular morphogenesis. Surprisingly, two-dimensional morphology and behavior of Dictyostelium amoebae lacking myosin I1 (mhcA-) is not dramatically altered when cells are examined using conventional imaging techniques. We have observed amoebae from the side (side-view microscopy or SVM) and find that wild-type but not mhcA-cells undergo extensive three-dimensional (3D) shape changes. These shape changes often occur above the plane of the substrate and are morphologically distinct from pseudopods. For example, unlike pseudopods, vertically extended cell regions are not enriched in F-actin and do not exclude organelles. In contrast, mhcA-cells generate F-actinfilled pseudopods and spread laterally but do not undergo vertical extension. When wild-type cells are removed from the substrate and shaken in suspension, they retain their irregular three-dimensional shape. Suspended mhcA-cells, however, rapidly become spherical. Thus, cells lacking myosin appear to be incapable of generating or maintaining 3D shape independent of a substrate. When a substrate is available, these cells can attach and spread on the surface in a myosinindependent manner. These previously undescribed defects of mhcA-cells reveal that 3D cell shape generation requires myosin I1 and is mechanistically distinct from pseudopod formation.