In order to determine the functional roles of the conserved sequence (NXNSSRFG) of the "switch I" loop (residues 233-240 in Dictyostelium myosin II), alanine scanning mutagenesis was performed on Dictyostelium myosin II. N233A and S237A mutant myosins did not bind a fluorescent analog of ADP, mant-deoxyADP, at the low concentration range (micromolar and had low level of ATPase activities. They were nonmotile when examined by the in vitro motility assay. Dictyostelium cells expressing these myosins showed worse phenotypes than that of myosin-null cells. In contrast to these mutant myosins, R238A myosin tightly bound mant-deoxyADP. However, the mutant had a defect in the ATP hydrolysis step and exhibited the lowest ATPase activities among the mutants examined here. The R238A myosin was nonmotile. R238C or R238H mutations, which mimic the Usher syndrome mutations, generated myosins with similar functional defects to those of the R238A mutation. Cells expressing the R238A myosin exhibited the phenotype similar to that of the myosin-null cells. N235A, S236A, F239A, and G240A myosins retained moderate levels of ATPase activities and could drive sliding of actin filaments at various speeds. Phenotypes of cells expressing them were very similar to that of the wild-type cells. Taken together, these results suggest that side chains of N233 and S237 may play essential roles in holding a nucleotide in the ATPase pocket and that R238 may play crucial roles in the ATP hydrolysis step, while those of the other residues in the switch I loop are not essential for the process.