A survey of adult patients was conducted in February 2006 to May 2006 in acute hospitals across England, Wales, Northern Ireland and the Republic of Ireland to estimate the prevalence of healthcare-associated infections (HCAIs). A total of 75 694 patients were surveyed; 5743 of these had HCAIs, giving a prevalence of 7.59% (95% confidence interval: 7.40-7.78). HCAI prevalence in England was 8.19%, in Wales 6.35%, in Northern Ireland 5.43% and in the Republic of Ireland 4.89%. The most common HCAI system infections were gastrointestinal (20.6% of all HCAI), urinary tract (19.9%), surgical site (14.5%), pneumonia (14.1%), skin and soft tissue (10.4%) and primary bloodstream (7.0%). Prevalence of MRSA was 1.15% with MRSA being the causative organism in 15.8% of all system infections. Prevalence of Clostridium difficile was 1.21%. This was the largest HCAI prevalence survey ever performed in the four countries. The methodology and organisation used is a template for future HCAI surveillance initiatives, nationally, locally or at unit level. Information obtained from this survey will contribute to the prioritisation of resources and help to inform Departments of Health, hospitals and other relevant bodies in the continuing effort to reduce HCAI.