During a 2-month period in 2005, 13 laboratories participated in a surveillance study of Clostridium difficile -associated disease (CDAD) in 17 hospitals in The Netherlands. The median incidence rate of CDAD was 16/10 000 patient admissions (2.2/10 000 patient-days) and varied from 1 to 46/10 000 patient admissions according to hospital. In total, 81 patients with CDAD were reported; 49 (61%) patients had nosocomial CDAD, and 29 (36%) patients were admitted to hospital when already suffering from diarrhoea. Two (2%) deaths were attributable to CDAD; both of these patients were admitted with severe community-onset CDAD and were aged >80 years. Among 64 toxinogenic isolates, ten (16%) belonged to PCR ribotype 027 and ten (16%) to PCR ribotype 014. Type 027 was identified in ten patients from one hospital during an unrecognised outbreak. Toxinotyping of the 64 isolates revealed the presence of six different toxinogenic types, with 41 (64%) isolates of toxinotype 0, ten (16%) isolates of toxinotype III, and nine (14%) isolates of toxinotype V. Of the 64 toxinogenic isolates, seven (11%) had a 39-bp deletion in the tcdC gene, 11 (17%) had an 18-bp deletion, and one (1%) had a deletion of c. 44 bp. Genes for binary toxin were present in 21 (33%) of the 64 toxinogenic isolates, mainly associated with toxinotypes III and V. It was concluded that the median CDAD incidence rate of 16/10 000 patient admissions in The Netherlands is considerably lower than that in Canada and the USA, and that the emerging type 027 can spread unnoticed. The high proportion (36%) of CDAD cases with a community onset has important implications for future studies of the epidemiology of CDAD.