A prevalence study of hospital-acquired infections (HAI) was carried out in 14 of 112 Greek hospitals (15.7%), scattered throughout Greece. Five of seven Greek university hospitals and nine regional hospitals participated in the one-day study, and 3925 hospitalized patients (10.5% of the total hospital beds in Greece) were recorded. The aim of this project was to organize a surveillance of HAI with the participation of the greatest possible number of Greek hospitals, transferring the experience from the local Cretan infection control network in an effort to create a nationwide network. Special attention was paid to recruit all Greek university hospitals in our attempt to expand the study base. Co-ordination of the participating centres, education of the infection control teams on surveillance methods, preparation of agreed definitions, and elaboration of the protocol for the collection of the data were the major objectives of this study. The difficulties, however, were limited resources and the lack of skilled personnel. The overall prevalence of HAI was found to be 9.3%. The most common HAI recorded involved lower respiratory tract infections (30.3%), followed by urinary tract infections (22.7%), bloodstream infections (15.8%), and surgical site infections (14.8%). The greatest prevalence rate was found in the adult ICU (48.4%), followed by the neonatal ICU (30.3%). The duration of hospitalization, the number of operations, the total number of used devices and invasive procedures were significantly correlated with HAI. Positive cultures were found in 51.5% of the cases. The most frequently isolated micro-organisms were: Pseudomonas aeruginosa (16.6%), Escherichia coli (10.8%), Klebsiella pneumoniae (10.3%), Staphylococcus epidermidis (8.1%) and Staphylococcus aureus (7.6%). The administration of antibiotics was also recorded. The prevalence of antibiotic use was 51.4%.