T lymphocyte-mediated immunity is important for resistance to Francisella tularensis. To characterize the specificity of this immunity, we used membrane proteins and two lipopolysaccharide (LPS) preparations. Both membrane proteins were heat-modifiable, as indicated by their migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). One had an apparent molecular mass (Mm) of 120 kilodaltons (kDa) when solubilized in the SDS buffer at room temperature, but 17 kDa after heating. The respective values for the other protein were 35 kDa before and 40 kDa after heating. Both proteins were purified by a preparative SDS-PAGE. The LPS-containing preparations were isolated by aqueous phenol (WP) or PCP (phenol-chloroform-petroleum ether) extraction (LPS-R), and rendered protein-free by treatment with proteinase K. Lymphocytes from nine subjects immunized with a live tularemia vaccine from one to three years earlier responded specifically to both an F. tularensis whole cell antigen and the 17 kDa protein in the lymphocyte blast transformation test. By contrast, the 40 kDa protein and the two LPS preparations did not stimulate any detectable lymphocyte proliferation.