The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.