A mutagenized subclone of the murine EL-4 thymoma (clone B5) is approximately 30 times more potent than parental EL-4 cells in stimulating proliferation and Ig secretion of murine or human B cells by direct cell contact in the presence of appropriate cytokines. In this study we found that CD40 ligand (CD40L) expression was constitutive and very similar on EL-4 B5 and parental EL-4 cells according to Northern blot and flow cytometry. Activation with phorbol 12-myristate 13-acetate (PMA) alone, PMA and ionomycin, interleukin-1 (IL-1) or human T-cell supernatant did not lead to significant CD40L up-regulation. A receptor-binding assay with soluble CD40 did not reveal different ligand affinities. However, murine and human soluble CD40-IgFc fusion proteins inhibited human B-cell stimulation by EL-4 B5 cells in the presence of human T-cell supernatant. Inhibition was 96% when soluble CD40 was added on day 0 of culture and progressively decreased when the CD40 was added subsequently. Ig secretion by cytoplasmic Ig-positive cells was no longer inhibited. These findings imply that, although CD40 ligand is necessary for B-cell activation by EL-4B5 cells, additional molecule(s) must be responsible for the increased helper activity of the EL-4 B5 clone.