Abstract Evidence is presented for the physiological role of the phosphoenolpyruvate:lactose phosphotransferase system in the utilization of lactose by Staphylococcus aureus. The concept is that this organism takes up lactose (and other sugars) by group translocation, i.e. the sugar is phosphorylated while it traverses the cell membrane, and that the phosphotransferase system is responsible for this process. A study of mutants defective in proteins of the phosphotransferase system provided evidence to support this interpretation. Mutants lacking Enzyme I were unable to ferment variety of sugars, and could not take up methyl α-glucopyranoside (an analogue of glucose) or thiomethyl β-galactopyranoside (an analogue of lactose). Mutants defective in Enzyme IIlac or Factor IIIlac of the phosphotransferase system were also incapable of transporting thiomethyl β-galactopyranoside, but transported methyl α-glucopyranoside at normal rates. Since the mutants were derived from constitutive strain, the inability of the Enzyme IIlac (or Factor IIIlac) mutants to transport lactose or its analogues cannot be explained by lack of induction of the lactose operon. The mutants did not catalyze facilitated diffusion process, therefore indicating that the phosphotransferase system is responsible for translocation per se, and that it does not act as a sugar trap. Preliminary evidence suggests that galactose 6-phosphate is metabolized in S. aureus by conversion to galactonic acid 6-phosphate.