Supernatants from S26.5 helper T cells, autoimmune viable motheaten (mev/mev) mouse spleen cells, EL4 lymphoma cells, and recombinant DNA-derived interferon gamma (IFN-gamma), all of which display B cell maturation factor (BMF) activity, were assayed for effects on B cell proliferation alone and with Dextran Sulfate (DxS) and anti-immunoglobulin antibodies (alpha-Ig). Both EL4 and S26.5 supernatants showed BCGF-II (DxS co-stimulator) activity, whereas only EL4 supernatant had BCGF-I (alpha-Ig co-stimulator or BSF-I) activity. Supernatants from mev/mev spleen cells and recombinant DNA-derived IFN-gamma showed no activity in either assay. Fractionation of S26.5 supernatant by chromatofocusing showed a divergence of BMF activity (BMF-T, pIa of 6.0) from BCGF-II activity (pIa of 5.4), providing evidence for their physical nonidentity. IFN-gamma, which decreases B cell viability in culture, was separable from BMF-T by phenyl-Sepharose chromatography. BMF-T from S26.5 supernatant was separated from IFN-gamma and BCGF-II and was shown to induce B cell maturation without affecting B cell proliferation. The molecular characteristics of the purified BMF-T were pIa 6.0, Mr 55,000 by G-75 gel filtration, and Mr 16,000 by SDS-PAGE. These data demonstrate that several lymphokines (BMF) exist that mediate the maturation of B cells to active Ig secretion without stimulating B cell proliferation.