Clostridium difficile is isolated from the intestinal tracts of > 50% of healthy infants. The mechanism by which intestinal colonisation of infants by toxigenic C. difficile is generally asymptomatic is unknown but may reflect the presence in human milk of neutralising activity against C. difficile toxin A. On this basis, the ability of human milk to inhibit the binding of toxin A to a purified hamster brush border membrane receptor was determined. Ten milk samples from healthy volunteers in various stages of lactation inhibited the binding of toxin A to the receptor by an average of 90%. Heating and dialysis did not significantly alter the inhibitory activity of any of the milk samples. Human milk protected adult hamsters against a lethal challenge with toxin A but had no effect on the cytotoxic activity of the toxin. SDS-PAGE and ligand blot analyses showed that there were at least four distinct factors in human milk that specifically bound toxin A. Thiophilic adsorption chromatography was used to separate immunoglobulin from non-immunoglobulin components of human milk. IgA was the only immunoglobulin detected in human milk and > 90% of this immunoglobulin was recovered after purification by thiophilic adsorption. Both the unbound non-immunoglobulin and bound immunoglobulin fractions of human milk inhibited the binding of toxin A to the purified receptor. These results suggest that human milk may be important in protecting infants against C. difficile-associated intestinal disease.