Duodenitis-proximal jejunitis (DPJ) is an idiopathic condition in the horse characterized by inflammation and oedema of the duodenum and proximal jejunum. Clinical signs include colic, ileus, depression, fluid accumulation in the small intestine and stomach, and endotoxaemia. The objective of this study was to investigate prospectively the role of Clostridium difficile in this idiopathic disease. Nasogastric reflux from 10 consecutive cases with DPJ and 16 consecutive horses with other causes of nasogastric reflux was cultured for C. difficile, other Clostridium spp., and Salmonella. Toxigenic strains of C. difficile were isolated from 10/10 (100 %) of horses with DPJ and 1/16 controls (P<0·0001). No other known pathogenic clostridia were isolated from either group. Results of this study suggest that C. difficile might be an important cause of this syndrome.