Vacuolar-type H(+)-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (similar to 70 s(-1)) with the K(m) values for ADP and phosphate being 4.7 +/- 0.5 and 460 +/- 30 mu M, respectively. Single molecule observation showed V-ATPase rotated in a 120 degrees stepwise manner, and analysis of dwelling time allowed the binding rate constant k(on) for ATP to be estimated (similar to 1.1 x 10(6) M(-1) s(-1)), which was much lower than the k(on) (= V(max)/K(m)) for ADP (similar to 1.4 x 10(7) M(-1) s(-1)). The slower kon ATP than kon ADP and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.