Phytochrome extracted from shoots of dark-grown rye (Secale cereale cv Rymin) and oat (Avena sativa cv Garry) as the far-red-form (Pfr) and/or under conditions conducive to oxidation exhibited a blue shift in the visible absorption maximum of its red-light-absorbing form (Pr) relative to that measured in vivo. This spectral alteration could not be reversed but could be prevented by inclusion of 10 millimolar diethyldithiocarbamate and 140 millimolar 2-mercaptoethanol in homogenization buffers. Similar blue shifts were induced in purified rye phytochrome by addition of the sulfhydryl-modifying reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In spectrally normal phytochrome (i.e., no detectable blue shift), Pfr had three to four more sulfhydryls available for rapid reaction with DTNB than did Pr. This difference was maintained over a 2.5-hour time course. Phytochrome purified under conditions resulting in a blue-shifted Pr absorption maximum exhibited a decreased short-term reactivity of Pfr to DTNB. Comparison of the binding and elution of altered and unaltered phytochrome from agarose-immobilized Cibacron blue 3GA confirmed that the Pfr form of spectrally normal phytochrome had a greater affinity for the dye than did the Pr form but that spectral alteration of phytochrome was accompanied by a loss of this difference as evidenced by an increased binding of Pr to the dye. It was concluded that phytochrome has highly reactive sulfhydryl residues located on the portion of the protein that undergoes conformational changes on interconversion of Pr and Pfr and that these residues require rigorous protection in order to extract the native form of the protein from plant tissue.