Extraction of Dictyostelium amoebae with Triton X-100 produces robust cytoskeletons composed mainly of actin and myosin II. These cytoskeletons rapidly contract when mixed with Mg-ATP in simple buffers. The Triton-soluble fraction was found to contain a GTP-dependent activity that prevented contraction by Mg-ATP. This activity was purified, and identified, as nucleoside diphosphate kinase (NDP kinase). The apparent inhibition resulted from pre-contraction of the cytoskeletons. Tightly bound cytoskeletal ADP was presumably phosphorylated, and the resulting ATP powered contraction. NDP kinase appeared to be unique in this capacity, since other regenerating systems did not cause pre-contraction. Reconstitution experiments demonstrated that the kinase must be in physical contact with the cytoskeleton. These results suggest that Dictyostelium NDP kinase is able to channel ATP to the myosin molecule, and this could play a role in directly regulating cytoskeletal contraction or in facilitating contraction under conditions where intracellular ATP concentrations are low. This ability to modulate cytoskeletal contraction could help to explain observations in other systems whereby defects in NDP kinase result in abnormal development or changes in the metastatic potential of cancer cells.