The effect of sulphide on resting oxidized cytochrome c oxidase was studied by both e.p.r. and optical-absorption spectroscopy. Excess sulphide causes some reduction of cytochrome a, CuA and CuB, and the formation of the cytochrome a3-SH complex after about 1 min. After several hours in the presence of excess sulphide only the e.p.r. signals due to low-spin ferricytochrome a3-SH persist, giving a partially reduced species. Re-oxidation of this partially reduced sulphide-bound enzyme by ferricyanide makes all of the metal centres except CuB detectable by e.p.r. We conclude that sulphide has reduced and binds to CuB as well as to ferricytochrome a3. Sulphide binding to cuprous CuB may raise its mid-point potential and make re-oxidation difficult. Addition of reductant (ascorbate + NNN'N'-tetramethyl-p-phenylenediamine) and sulphide together to the oxidized resting enzyme produces a species in which cytochrome a and CuA are nearly completely reduced and cytochrome a3 is e.p.r.-detectable as approx. 80% of one haem in the low-spin sulphide-bound complex. The g = 12 signal of this partially reduced derivative is almost unchanged in magnitude relative to that of the resting enzyme; this suggests that the g = 12 signal may arise from less than 20% of the enzyme and that it may be relatively unreactive to both ligation and reduction. Such a reactivity pattern of the g = 12 form of the oxidase is also demonstrated with the ligands F- and NO, which are thought to bind to cytochrome a3 and CuB respectively.