N-Methylhydantoin amidohydrolase, which catalyzes ATP-dependent hydrolysis of N-methylhydantoin to N-carbamoylsarcosine, was found to hydrolyze several nucleoside triphosphates tee nucleoside diphosphates not only in the presence but also in the absence of amide substrates. Amide substrates, such as N-methylhydantoin and dihydrouracil, seem to be absolutely necessary for hydrolysis of ATP and dATP. However, N-methylhydantoin inhibited the hydrolysis of nucleoside triphosphates other than ATP and dATP, The kinetic data suggest that the presence of an amide substrate changed the affinity of the enzyme toward nucleoside triphosphates.