Between January and April 1993, culture for Clostridium difficile and a faecal cytotoxin assay were performed on 500 selected specimens. Isolates from culture-positive patients from whom faecal samples were cytotoxin negative were also examined in vitro for cytotoxin production. The significance of a positive culture result in the absence of faecal cytotoxin was assessed. Forty-one of the 500 specimens were toxin positive. In only 25 of these was Clostridium difficile examination specifically requested. Six of nine culture-positive cytotoxin-negative patients (11 specimens) had recently received antibiotics. In four of these, Clostridium difficile was considered to be of possible clinical significance. Culture and in vitro determination of toxin production of isolates may aid in the diagnosis of some additional cases, but cytotoxin detection remains the single optimal routine laboratory method for diagnosis.