The destructive course of Staphylococcus aureus arthritis is due to certain leukocytes and their products, mainly cytokines. The cellular source of cytokines mediating this inflammatory process has not been previously assessed on a protein level. Using a mouse model of hematogenously induced S. aureus infection the intracellular production of IFN-gamma, IL-4 and IL-10 in splenic B and T cells have been determined. This has enabled us to define distinct Th1 vs Th2 and Be1 vs Be2 populations of lymphocytes participating in S. aureus infection. Spleen cells were obtained before, and at different time intervals during the first week of infection and re-stimulated in vitro with staphylococcal peptidoglycan, formalin killed staphylococci and TSST-1. The different antigens used for re-stimulation gave rise to different cytokine profiles in analysed T cells (identified as CD4+) and B cells (identified as CD19+). TSST-1 acted as the most potent re-stimulator and we found that 40% of the CD4+ cells responded with IFN-gamma production, and unexpectedly almost 20% of the CD19+ cells. As to IL-4 and IL-10 production, the percentage of B cells expressing these cytokines was higher than the percentage T cells and the peak of their appearance appeared later than that of IFN-gamma. This finding indicates that Be1 cells are an important source of IFN-gamma early during the infection and that the production of the Th2 cytokines in B cells downregulates its production of IFN-gamma. In conclusion this study shows that both B and T cells contribute to the cytokine production during S. aureus infection in a complex pattern.