In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S-3 State undergoes biphasic kinetics although the fast phase could not be fully resolved at that time Flessinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci, U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O-2 as a function of time after injection of (H2O)-O-18 into spinach thylakoid samples preset in the S-3 State at 10 degrees C. For measurements made at m/e = 34 (i.e., for the mixed labeled O-16,18(2) product), the two kinetic components are observed: a slow component with k(1) = 2.2 +/- 0.1 s(-1) (t(1/2) similar to 315 ms) and a fast component with k(2) = 38 +/- 4 s(-1) (t(1/2) similar to 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e,, for the double labeled O-18,18(2) product), only the slow component (k(1)) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled O-18,18(2) product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k(1) increases by a factor of 10 over the range 0-20 degrees C while k(2) increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol(-1) for the slow component and 39 +/- 5 kJ mol(-1) for the fast component. The different kinetic components in the O-18 isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S-3 state, prior to the O-2 release step (t(1/2) similar to 1 ms at 20 degrees C). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.