B cell-activating factor (BCAF) has been characterized and partially purified from the supernatant of the murine tumor T cell line 373. BCAF has an apparent molecular mass of 15 to 20 kDa when analyzed by Superose 12 fast protein liquid chromatography (FPLC) gel filtration and a pI of 4.5 to 5.0 when analyzed by FPLC chromatofocusing. Concentrated supernatant was applied to a nickel chelating column and unadsorbed active material was further purified by two sequential C4 reverse-phase high-performance liquid chromatography steps. This purification procedure allowed a complete separation of interleukin 2 and interleukin 4 activities from BCAF. This partially purified BCAF induces Ia expression, cell size increase and proliferation of small resting B cells. Furthermore, we have shown that the activity of partially purified BCAF is insensitive to treatment by monoclonal antibodies specific for interleukin 4 and interleukin 5.