Solubilized constituents from Listeria monocytogenes were fractionated by various techniques including isopycnic gradient centrifugation, molecular sieve chromatography, and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Fractionated material was tested in vitro for mitogenic and antigenic activity by quantitating the proliferation of splenic lymphocytes and the interleukin production by peritoneal T cells. Fractionation by isopycnic gradient centrifugation revealed both antigenic and mitogenic material fractionating with the protein at a density of 1.3 g/ml. This characteristic density, together with the reduction of activity with trypsin treatment, defined the material as protein. This material was termed soluble listerial proteins (SLP). Fractionation of SLP by molecular sieve chromatography using Sephacryl 200 (S-200) revealed predominant antigenic and mitogenic activity in proteins of greater than 100,000 m.w. In contrast, fractionation of SLP by preparative SDS-PAGE (nonreducing conditions) showed activity in groups of proteins with m.w. of less than 76,000. This difference (S-200 vs SDS-PAGE) may indicate an aggregation or subunit composition which is disrupted by SDS. When fractionated by SDS-PAGE, antigens which induced macrophage-dependent interleukin production by Listeria-immune T cells were observed over a broad range of molecular sizes. Major groups of antigenic proteins were observed at 57,000 to 76,000 m.w., approximately 40,000 and less than 25,000 m.w. Mitogenic activity (spleen cell proliferation) was associated with a more restricted group of proteins with major peaks at 57,000 and 40,000 m.w., with some weak activity in proteins less than 20,000 and greater than 64,000 m.w. Experiments involving T or B lymphocyte-depleted spleen cells and spleen cells from athymic mice revealed that the mitogenic response of splenic lymphocytes to SLP was predominantly B cell-mediated. Thus, we have defined groups of listerial proteins with potent antigenic activity with respect to T lymphocyte activation and as mitogenic activity for B cells.