Background: The phosphoenolpyruvate (PEP):lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICB<sup>Lac</sup>). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. Results: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-β-D-galactopyranoside and EIICB<sup>Lac</sup> with UV light caused a loss of EIICB<sup>Lac</sup> activity. Nevertheless, photoinactivated EIICB<sup>Lac</sup> could still be phosphorylated with [<sup>32</sup>P]PEP. Proteolysis of photoinactivated [<sup>32</sup>P]P-EIICB<sup>Lac</sup> with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245–247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [<sup>32</sup>P]P∼EIICB<sup>Lac</sup>, photoinactivated [<sup>32</sup>P]P-EIICB<sup>Lac</sup> remained strongly phosphorylated. Conclusion: Phosphorylation of the –OH group at C6 of p-nitrenephenyl-β-D-galactopyranoside covalently bound to EIIC<sup>Lac</sup> by the histidyl-phosphorylated [<sup>32</sup>P]P∼EIIB<sup>Lac</sup> domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-β-D-galactopyranoside into the active site of modelled EIIC<sup>Lac</sup> suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.