The amounts of ATP and ADP bound to 21S dynein during the ATPase reaction were measured in the presence of 2.83 mg/ml 21S dynein, 2 mM PEP, 4 mg/ml PK, 0.1 M KCl, 5 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 50% [2-3H]glycerol, and 20 mM imidazole at pH 7.0 and 0 degrees C. The maximum amounts of ATP and ADP bound to 21S dynein were 0.29 and 0.55 mol/(10(6) g protein), respectively. The dissociation constants of ATP for the ATP and ADP binding (4 microM) were almost equal to the Km value (3.7 microM) of dynein-ATPase in the steady state. The amount of bound ADP during the initial phase showed an overshoot, which reached 0.6-0.8 mol/10(6) g protein at 5 s, then decreased to the steady state level within 20 s. Furthermore, the rate of TCA-Pi liberation during the initial 5 s was 6 times the steady-state rate. The apparent Pi-burst size, estimated by extrapolating the steady-state Pi liberation to zero time, was 1.33 mol/(10(6) g protein). The true Pi-burst size was calculated to be 1.56 mol/(10(6) g protein) by correcting for the effect of Pi liberation at steady state. All these findings could be explained quantitatively by the following reaction scheme for 21S dynein ATPase in the presence of glycerol: (formula; see text) where K1 = 25.5 microM, and k2, k3, and k4 were 0.39, 0.21, and 0.11 s-1, respectively.