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Covalently bound pH sensitive dyes are an important tool for characterizing the proteolytic reactions of protein complexes that play key roles in biological energy transduction. Here we demonstrate the feasibility of this method for photosynthetic reaction centers (RCs) for the first time, by the highly selective attachment of two thiol reactive derivatives of fluorescein to the two H subunit cysteines of the photosynthetic RC from Rhodobacter sphaeroides R-26 The pK(a) shifts of the dyes upon binding to the protein and in response to high salt were measured, and interpreted based on the structure of the RC. 2-[(5-fluoresceinyl)aminocarbonyl]ethyl-methanethiosulfonate was attached to Cys H156 and fluorescein-5-maleimide to Cys H234. By following the absorption changes of bound fluorescein (500 nm), and those of the hydrophilic pH indicator 8-hydroxypyrene-1,3,6-tris-sulfonic acid (468 nm), the surface and bulk pH were monitored separately with less than 5% crosstalk. Flash-induced proton uptake and external calibrations by mixing with aliquots of acid were measured in different redox states of the RCs. The results indicate that the charge in the quinone acceptor complex after flash activation (primary quinone acceptor (Q(A))- or secondary quinone acceptor (Q(B))-) has no effect on the surface pH and potential in the vicinity of these two attachment sites, between pH 6.5 and 9. Application of the method to other surface locations is discussed.