Cytochrome c oxidase is a redox-driven proton pump. The enzyme has two proton input pathways, leading from the solution on the N-side to the binuclear center. One of these pathways, the K-pathway, is used for proton uptake upon reduction of the binuclear center. It is also important for local charge compensation during reaction of the fully reduced enzyme with O2. Two different locations have been proposed to constitute the entry point of the K-pathway: near S(I-299) or near E(II-101), respectively, in the Rhodobacter sphaeroides enzyme. The experiments discussed in this study are aimed at identifying the location of the entry point. The kinetics and extent of flash-induced proton release coupled to oxidation of heme a3 (tau congruent with 2 ms at pH 8.8 in the wild-type enzyme) in the absence of O2 were investigated in the ED(II-101), SD(I-299), and KM(I-362) mutant enzymes, i.e., at the two proposed entry points and in the middle of the pathway, respectively. This reaction was completely blocked in KM(I-362), while it was slowed by factors of 25 and 40 in the ED(II-101) and SD(I-299) mutant enzymes, respectively. During reaction of the fully reduced enzyme with O2, electron transfer from heme a to the catalytic site (during P(R)-formation) was blocked in the KM(I-362) and SD(I-299)/SG(I-299) but not in the ED(II-101)/ EA(II-101) mutant enzymes. The results are interpreted as follows: Residue K(I-362) is involved in both proton transfer and charge compensation (in different reaction steps). The impaired proton release in the S(I-299) mutant enzymes is an indirect effect due to an altered environment of K(I-362). E(II-101), on the other hand, is likely to be part of the K-pathway since mutation of this residue results in impaired proton release but does not affect the P(R) formation kinetics; i.e., the properties of K(I-362) are not altered. Consequently, we conclude that the entry point of the K-pathway is located near E(II-101).