The coincidental increase in norovirus outbreaks and Clostridium difficile infection (CDI) raised the question of whether these events could be related, e.g. by enhancing spread by diarrhoeal disease outbreaks. Therefore, we studied the prevalence of C. difficile in outbreaks of viral gastroenteritis in nursing homes for the elderly and characterised enzyme immunoassay (EIA)-positive stool samples. Stool samples from nursing home residents (n=752) in 137 outbreaks of viral aetiology were investigated by EIA for the presence of C. difficile toxins. Positive samples were further tested by a cell neutralisation cytotoxicity test, a second EIA and culture. Cultured isolates were tested for the presence of toxin genes, the production of toxins and characterised by 16S rRNA polymerase chain reaction (PCR) and sequencing. Twenty-four samples (3.2%) tested positive in the EIA. Of these 24 positive samples, only two were positive by cytotoxicity and three by a second EIA. Bacterial culture of 21 available stool samples yielded a toxinogenic C. difficile PCR ribotype 001 in one patient sample only. In conclusion, we found no evidence in this retrospective study for an association between viral gastroenteritis outbreaks and C. difficile. The high rate of false-positive EIA samples emphasises the need for second confirmation tests to diagnose CDI.