Toxins A and B are known to be the primary virulence factors of Clostridium difficile. Other potential virulence factors have been identified such as binary toxin (actin-specific ADP-ribosyltransferase toxin, or CDT). A retrospective case-control study was performed in order to identify clinical features and risk factors of C. difficile-associated diarrhoea due to binary toxin-producing strains. Each case (a patient with diarrhoea due to binary toxin-producing strain) was compared with two controls (patients with diarrhoea due to a C. difficile strain that did not produce binary toxin) matched for ward and date of hospitalization. cdtA and cdtB genes were screened by PCR. Production of CDT was studied by Western blotting using an antiserum against Ia and Ib from the Clostridium perfringens iota toxin, and the activity of the binary toxin was assessed using an ADP-ribosyltransferase assay. Twenty-six cases (14 males and 12 females) were identified in 1999 and 2000. Cases and controls did not differ significantly for sex, age, previous administration of antibiotics or frequency of endoscopic examination. Diarrhoea was community-acquired more often in cases than in controls (65.4 vs 35.7 %, P = 0.017) and more often represented the cause of hospitalization (61.5 vs 26.2 %, P = 0.003). Moreover, diarrhoea in cases was more frequently associated with abdominal pain (63.6 vs 39.4 %, P = 0.07) and with liquid stools (76.9 vs 59.5 %, P = 0.14) than in controls. These results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea.