Protein A (PA), a cell wall component of SAC, activates human B cells by cross-linking the Fabs of membrane immunoglobulins. Recent data indicate that PA binds only to Fabs that use VH3 heavy chains, and thus it has been designated as a B-cell superantigen. We previously reported that Staphylococcus aureus Cowan I (SAC)-induced IgM rheumatoid factor (RF) by human PBMC was mediated by PA. Therefore, we sought to determine if SAC-induced IgMRF production was confined to PA-binding B cells and if these B cells were enriched for the expression of VH3 heavy chains. We observed that the elicitation of IgMRF in response to SAC was limited to a subset of B cells that bind PA and that this subset was enriched for VH3 mRNA expression. Taken together, these results suggest that IgMRFs produced in response to SAC are enriched for usage of VH3 heavy chains. Thus, this SAC-induced autoantibody response appears to represent a new B-cell superantigenic property of PA.