AbstractTo study the in vivo role of myosin‐II inAcanthamoeba castellanii, motile cells were microinjected with monoclonal antibodies raised against the myosin‐II heavy chain. All injected cells underwent a transient shock response. It was found that although injection of buffer alone or of an endogenousAcanthamoebaprotein decreased the motility of injected cells from 7 μm/min to ∼3 μm/min, injection of monoclonal antibodies specific for myosin‐II decreased motility further to ∼0.8 μm/min. This effect was seen whether or not the monoclonal antibody to myosin‐II inhibited the actomyosin‐II MgATPase activity in vitro. Levels of antibody far in excess of endogenous myosin‐II concentrations could not completely block amoeboid movement. The morphology of moving antimyosin‐II‐injected cells was unusual, suggesting a greater defect in the ability to retract the trailing edge of the cell rather than to extend the leading edge. Endosomes frequently disappeared from injected cells, and although buffer‐injected cells rapidly recovered visible endosomes (50% recovery at 5 min), endosomes were not seen in antimyosin‐II‐injected cells until, on the average, ∼50 min after injection. Injection of a nonspecific antibody or of a nonspecific exogenous protein (ovalbumin) also decreased the mobility of the injected cells beyond that of buffer‐injected cells (to ∼1 μm/min). These cells tended to recover endosomes more rapidly (∼25 min) than cells injected with antimyosin‐II monoclonal antibodies. The inability of antibodies to myosin‐II to inhibit completely any of the movements studied suggests that although myosin‐II probably plays a role in these motilities, the cell either routinely uses or can draw upon another cytoplasmic motor to maintain locomotion, organelle movement, co