Three threonine residues in the tail region of Dictyostelium myosin II heavy chain have been implicated previously in control of myosin filament formation. Here we report the in vitro and in vivo consequences of converting these sites to alanine residues, which eliminates phosphorylation at these positions, or to aspartate residues, which mimics the negative charge state of the phosphorylated molecule. Alanine substitution allows in vitro assembly and in vivo contractile activity, although this myosin shows substantial over-assembly in vivo. Aspartate substitution eliminates filament assembly in vitro and renders the myosin unable to drive any tested contractile event in vivo. These results demonstrate that heavy chain phosphorylation plays a key modulatory role in controlling myosin function in vivo.