Staphylococcus aureus is an important human and animal pathogen that harbors protein kinases. The proteins phosphorylated in this bacterium grown on glucose minimal medium have been in vivo labeled with[(32)P]-orthophosphate and analyzed by two-dimensional gel electrophoresis followed by MS. A total of 11 glycolytic phosphoproteins have been identified and verified. In vitro analyses have revealed that phosphorylation of these glycolytic enzymes is catalysed primarily through the activity of an endogenous serine/threonine kinase and to a lesser extent by a tyrosine kinase. The identification of these phosphoproteins should prove helpful in understanding and unravelling of the role of phosphorylation with respect to pathogenesis and virulence in this organism.