Human IgM that express the variable region genes of the VH3 family bind staphylococcal protein A (SPA). We previously reported that the SPA-binding IgM can be divided into two groups based on the differential binding avidities for solid-phase SPA. To study the molecular basis for these differences, we cloned B cells from human blood by EBV transformation. The nucleotide sequences of the expressed Ig heavy chain genes were determined on 20 B cell clones that produce SPA-binding IgM. The germ-line VH3 gene usage in IgM with high avidities for SPA were distinct from the germ-line VH3 genes used in IgM with low avidities for SPA. There was no correlation in the usage of D or JH genes or in the usage of light chains in IgM according to the SPA binding avidity. These results suggest that the differential binding avidities for SPA are at least partly due to specific germ-line VH3 gene usage. An investigation of direct binding of SPA to the synthetic peptides corresponding to the portions of the variable regions of SPA-binding and non-SPA-binding IgM showed that the peptides corresponding to the VH3 family specific framework region 3 sequences had significant SPA binding capacities, while the peptides corresponding to the other subdomains and those corresponding to framework region 3 of the reported VH3 sequences from non-SPA-binding IgM showed little or no binding. It is of interest that the Ig-framework region 3 subdomain corresponds to the fourth hypervariable region, which in the TCR-beta chain has been implicated as a critical site for T cell superantigen binding.