Respiratory nitrate reductase with lactate as a hydrogen donor has been studied in cells and spheroplast preparations of wild type and heme-deficienct mutants of Staphylococcus aureus. The activity is rapidly induced when suspensions of aerobically grown cells are incubated without aeration in a complete medium with nitrate. In ruptured spheroplast preparations, the activity with lactate as the donor is located in the membrane fraction, whereas at least 50% of the activity assayed with reduced benzyl viologen is in the cytoplasm. The reductase is inhibited by azide and cyanide, and the lactate-linked system is also sensitive to oxamate, 2-heptyl-4-hydroxyquinoline-N-oxide, dicoumarol, and p-chloromercuribenzoate. An inactive form of the reductase is apparently made during induction with tungstate; this can be activated by subsequent incubation with molybdate in the presence of chloramphenicol. Nitrate reductase activity with reduced benzyl viologen as the donor is induced in suspensions of heme-deficient mutants in the presence or absence of heme. The proportion of cytoplasmic activity is increased in the absence of heme. The staphylococcal nitrate reductase has many of the characteristics commonly associated with the respiratory enzyme in other organisms, but the apparent predominance of cytoplasmic activity is unusual.