The production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated. Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells. Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay. Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle. The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed.