Synovial fluid from patients with rheumatoid arthritis (RA-SF) contains in vivo produced cytokines and inflammatory mediators, including a factor that induces IgG2b production of lipopolysaccharide (LPS) preactivated murine B lymphocytes. In order to determine the mechanism by which RA-SF acts on LPS activated mouse B cells, CBA/N mice were used as an experimental model. The X-linked immunodeficiency of these mice is caused by a point mutation in the Bruton's tyrosine kinase (btk) gene. We have earlier shown that RA-SF can reconstitute the CBA/N B cell deficiency in vitro and in vivo, with regard to IgG2b production after LPS stimulation. Since transforming growth factor (TGF)-beta has been suggested to be a switch factor for IgG2b, we aimed at investigating the role of TGF-beta in our experimental system. We found that TGF-beta could not mimic the effect of RA-SF on CBA spleen cells. A small increase of IgG2b secretion was observed with spleen cells from normal CBA mice, whereas Ig secretion of all isotypes was suppressed in CBA/N spleen cells treated with TGF-beta at any concentration. Neutralizing antibodies against TGF-beta suppressed the response of CBA B cells, whereas the response by CBA/N B cells was enhanced by the same antibody preparation. Here we also show that the abnormal B cell responsiveness to TGF-beta, typical of CBA/N, co-segregates with the btk mutation in male (CBA x CBA/N)F2 spleen cells. This was determined by allele specific PCR recognizing the identified base substitutions of the btk gene, typical of the two strains. We propose that RA-SF contains a factor, separate from TGF-beta, that is involved in the differentiation of IgG2b expressing cells.