The non-toxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent mucosal adjuvant and immunomodulator capable of blocking autoimmune disease. These effects are linked with its ability to modulate lymphocyte populations--a feature that is dependent on binding to ubiquitously expressed cell surface receptors. Here, we demonstrate that EtxB can trigger up-regulated expression of class II MHC and CD25 on purified populations of B lymphocytes, suggesting that EtxB can directly activate biochemical signalling pathways in these cells. The nature of the intracellular signalling events was investigated. B cells cultured with EtxB, but not a non-receptor binding mutant protein, EtxB(G33D), caused the activation of the extracellular signal-regulated kinase (Erk) forms of mitogen-activated protein (MAP) kinase in a process that was dependent on MAPK/Erk kinase (MEK), phosphoinositide 3-kinase (PI3-kinase) and protein kinase C (PKC), as determined by the use of specific inhibitors. PI3-kinase was critical not only in the activation of MAP kinase but also in the up-regulation of both class II and CD25. However, MEK inhibition only partially abrogated the EtxB-mediated up-regulation of MHC class II expression and did not affect CD25 expression--findings suggesting that additional pathways downstream of PI3-kinase are involved. A role for PKC in these processes was suggested by the finding that inhibitors of PKC completely blocked EtxB-mediated CD25 up-regulation. Thus, we have shown that receptor binding by EtxB triggers multiple signalling pathways in B cells that regulate the expression of key cell surface molecules.