The Health Protection Surveillance Centre (HPSC) established a group to produce national guidelines for Clostridium difficile in Ireland in 2006. A laboratory questionnaire was distributed to determine current C. difficile diagnostic practices. Twenty-nine out of 44 laboratories providing C. difficile diagnostic services to 34 hospitals responded. Twenty-five out of 29 (86%) laboratories processed specimens for C. difficile and four (13.8%) forwarded specimens to another laboratory. Sixteen laboratories (64%) processed specimens for other healthcare facilities. None routinely examined stool for C. difficile, seven (28%) examined specimens only when requested to do so and 18 (72%) used specific selection criteria, including testing all liquid stools (39%), all nosocomial diarrhoea (44%), specific clinical criteria (28%) and history of antibiotic therapy (22%). All tested stool directly for C. difficile toxin with a variety of enzyme immunoassays, with 24 (96%) detecting both toxin A and B and one detecting toxin A only. Three (12%) laboratories used cytotoxicity assays; none used polymerase chain reaction and six (24%) laboratories performed C. difficile culture but only under specific circumstances. Seven (28%) laboratories had isolates typed during outbreaks, but none had the facilities to do so on-site. The HPSC group will produce national recommendations for laboratory diagnosis, surveillance and management of C. difficile infection. Since there are marked differences in diagnostic practices throughout the country and no national reference laboratory, the implementation of these recommendations will have cost implications that will need to be addressed.