F1-ATPase from Bacillus subtilis (BF1) is severely suppressed by the MgADP inhibition. Here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. Measurements with a tryptophan mutant of BF1 indicated that the noncatalytic sites could bind ATP normally. Furthermore, the mutant BF1 that cannot bind ATP to the noncatalytic sites showed much lower ATPase activity. It was concluded that the cause of strong MgADP inhibition of BF1 is not the weak nucleotide binding to the noncatalytic sites but the other steps required for the activation.