The technique of using sodium dodecylsulfate-polyacrylamide gel electrophoresis fractionated antigens (Ag) transferred to nitrocellulose filters was adopted to analyse T cell responses to Salmonella enteritidis 11RX Ag. Employing in vitro proliferation assays with T cells from S. enteritidis 11RX-primed (BALB/c x C57BL/6J)F1 mice as the measure of T cell stimulation, we have identified Ag able to stimulate T cells in the regions containing 16, 24, 34 and 50-60 kDa proteins, with dominant Ag activity at about 16 kDa. These results were confirmed with long-term, Ag-specific L3T4+ T cell lines which responded to molecules in the same four Mr regions, suggesting that no selection by a single antigenic determinant had occurred during more than 3 months of in vitro culture, or that all the molecules which were stimulatory shared at least one antigenic determinant. Because the seven clones we examined responded only to 16 kDa molecules, the former alternative is the more likely. Standard immunoblot analysis indicated that these Ag also act as major B cell stimulating determinants. T cells of BALB/c mice, which are 5-10 times more resistant to S. enteritidis 11RX than C57BL/6J mice, showed the same pattern of reactivity as F1 mice whereas the major antigenic region for T cells of C57BL/6J mice was located between 50 and 60 kDa.