Yersinia pseudotuberculosis-derived mitogen (YPM) is the unique Gram-negative bacillary superantigen known. In order to identify the regions on the YPM molecule involved in its superantigenic activity, seven overlapping peptides of the entire YPM molecule were synthesized and tested to evaluate their effects on the YPM-induced proliferation of human peripheral blood lymphocytes. A peptide corresponding to the N-terminal amino acid sequence (1–23) was found to inhibit YPM-induced lymphocyte proliferation in a concentration-dependent manner. The N-terminal peptide was found to show no inhibition of the proliferation induced by the other superantigen (staphylococcal enterotoxin B) or the other T-cell mitogen pertussis toxin, indicating that the inhibition is specific to YPM-induced proliferation. Thus, we have identified the N-terminal region (1–23) of the YPM as one of the functional regions responsible for its superantigenic activity.