Clostridium difficile toxins A and B cause antibiotic-associated colitis. Whereas antigenic determinants specifying neutralisation of toxin A have been partially elucidated, those of toxin B remain unknown. To define antigenic determinants of toxin B, synthetic peptides were prepared for five linear sequences selected by computer analysis for putative T and B epitopes. Peptides spanning the carboxy terminal region (aa 2155-2283) were also selected because this region contains repetitive units thought to bind the toxin to cell receptors. Multiple antigenic peptides were synthesised by linking four peptide copies to a core of four lysine residues (tetraMAP). Outbred mice were given four doses of each tetraMAP by intraperitoneal injection and specific immunoglobulins G and A were measured by enzyme-linked immunosorbent assay (ELISA) in serum, ascitic fluid and faeces. All 14 MAPs induced strong IgG responses against the homologous peptide; peptides representing aa 2155-2179 and 2246-2270 induced the strongest responses, of 592 and 493 ELISA units, respectively-although, to a lower extent, all 14 MAPs induced serum and faecal IgA responses against the homologous peptide. All MAPs induced IgG1 and IgG2b subclasses, documenting their capacity to elicit Th2-dependent mucosal immunity. IgG anti-MAPs were assayed for reaction with native toxins A and B; most anti-MAPs recognised the toxins only weakly or did not recognise them. Antibodies against peptide representing aa 2168-2192 recognised both native toxin B (19 ELISA units) and toxin A (2 ELISA units). None of the antibodies neutralised cytotoxicity of either toxin in cell culture. In contrast, four MAPs (aa 2080-2095, 2168-2192, 2220-2244 and 2233-2257) inhibited cytotoxicity when mixed with toxin B before addition to cells; inhibition was mediated by a direct interaction with toxin B.