Glucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-N-acetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from N-acetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N-acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N-acetylglucosamine. This and the preferential incorporation of N-acetylglucosamine over glucose into cell wall material showed that N-acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.