Point prevalence surveys are useful in detecting changes in the pattern of healthcare-associated infection (HCAI). In 2004 the Hospital Infection Society was asked to conduct a third national prevalence survey, which included England, Wales, Northern Ireland and the Republic of Ireland. A similar but not identical survey was carried out in Scotland. Data were collected on standardised forms using Centres for Disease Control and Prevention definitions. This report considers associations with a wide range of risk factors for all HCAI and for four main categories. The overall prevalence rate of HCAI was 7.6% and increased significantly with age. All risk factors considered were associated with highly significantly increased risk of HCAI, except recent peripheral IV catheter and other bladder instrumentation use. Primary bloodstream infection (PBSI) was associated with antibiotic, central intravenous catheter and parenteral nutrition use. Pneumonia was associated with antibiotic, central catheter, parenteral nutrition use, mechanical ventilation and current peripheral catheter use. Surgical site infection was associated with recent surgery, antibiotic and central catheter use, mechanical ventilation and parenteral nutrition. Urinary instrumentation and antibiotic use were associated with urinary tract infection. Patients under a critical care medicine consultant had the highest prevalence of HCAI (23.2%). This report highlights those areas requiring attention to prevent HCAI, i.e. device-related infections such as PBSI (e.g. central catheters) and pneumonia (e.g. mechanical ventilation) and should influence protocols for future prevalence surveys of HCAI, e.g. the recording of risk factors at the time of assessment only is sufficient.