Different classes of tryptophan residues in sarcoplasmic reticulum calcium-ATPase were investigated with respect to their exposure to quenchers and sensitivity to high-affinity calcium binding to the ATPase. The charged quenchers, iodide and cesium, produced only slight quenching of ATPase fluorescence, whereas noncharged acrylamide and notably oxygen produced significant quenching. This finding gives support to the proposed location of most of the tryptophan residues of the ATPase in transmembrane domains of this protein (MacLennan et al., 1985, Nature 316r, 696-700). Among the different quenchers tested, oxygen quenching alone was sensitive to calcium binding to the ATPase, indicating that oxygen quenched tryptophan residues located in regions of the ATPase molecule which undergo conformational changes upon calcium binding. Time-resolved oxygen quenching data were analyzed with a recently described model that takes into account the existence of two different classes of emitters in the ATPase (Ferreira and Verjovski-Almeida, 1991, J. Lumin. 48, 430-434): a short-lived blue-shifted exponential component plus a long-lived red-shifted continuous lifetime distribution. Oxygen quenching of the single-exponential lifetime component was found to be insensitive to calcium, whereas quenching of the distributed lifetime component was significantly (ca 25%) enhanced by calcium binding. The different sensitivities of the two tryptophan classes to calcium binding to the ATPase are interpreted in terms of the proposed location of tryptophan residues in relation to the calcium transport sites in the ATPase molecule.