The α and β subunits of F 1 -ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the β subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the α subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the α subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the β subunit with Gln. The resultant mutant α 3 β 3 γ complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant α 3 β 3 γ complex which had been photolabeled with 2-N 3 -[8- 3 H]ATP revealed that ATP tethered to the noncatalytic binding site was hydrolyzed, indicating that a primitive catalytic ability was generated at the α subunit by the introduced Glu.