Resistance to every family of clinically used antibiotics has emerged, and there is a pressing need to explore unique antibacterial targets. Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of many Gram-positive bacteria. Because WTAs play an essential role in Staphylococcus aureus colonization and infection, the enzymes involved in WTA biosynthesis are proposed to be targets for antibiotic development. To facilitate the discovery of WTA inhibitors, we have reconstituted the intracellular steps of S. aureus WTA biosynthesis. We show that two intracellular steps in the biosynthetic pathway are different from what was proposed. The work reported here lays the foundation for the discovery and characterization of inhibitors of WTA biosynthetic enzymes to assess their potential for treating bacterial infections.