Incubation of [gamma-32P]ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F1) results in binding of substrate primarily in a single, very high affinity (KA = 10(12) M-1) catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10(6)-fold, that is, to Vmax rates (Cross, R.L., Grubmeyer, C., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12101-12105). For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. Recently, Bullough et al. (Bullough, D.A., Verburg, J.G., Yoshida, M., and Allison, W.A. (1987) J. Biol. Chem. 262, 11675-11683) reported that when 5 to 20 microM concentrations of nonradioactive ATP were added as a cold chase to an enzyme-substrate complex consisting of F1 and ATP bound to the high affinity catalytic site, hydrolysis of the chase was commensurate with the turnover rate of the enzyme, whereas the hydrolysis of bound ATP was considerably slower. These authors suggested that the high affinity catalytic site on F1 is not a normal catalytic site. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to Vmax rates following addition of 5 microM ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F1 is a normal catalytic site.