The objective of this study was to assess the incidence of nosocomial rotavirus gastroenteritis among children <2 years of age. We conducted a prospective active surveillance for acute gastroenteritis (AGE) in the pediatric wards of 3 representative hospitals in Valencia (Spain) from October 2006 to March 2007, among children between 1 and 23 months of age with acute diarrhea. Children were followed up for 3 days after discharge. We obtained clinical and demographic information from participants and tested their stool specimens for rotavirus. A total of 1576 children were hospitalized at the 3 hospitals and 1300 (82.5%) were followed up as the study cohort. In 69 children, AGE started 48 hours after admission and were considered nosocomial infections. In 35 of the 59 cases where stool samples were obtained, rotavirus (RV) was present (59%), and in 12 of them symptoms started after discharge. The accumulated incidence of nosocomial rotavirus disease during the study period was 2.8 cases per 100 inpatients (95% CI: 1.9-3.8), and the incidence rate was 4.8 cases per 1000 hospital days (95% CI: 3.2-6.5). The most commonly found genotype in nosocomial infection was G9P[8], in 23 cases (66%), followed by G1P[8] in 4 cases (11%). The total economic cost was 883 euro per case. Active surveillance demonstrated that the burden of nosocomial rotavirus disease is substantial, and G9P [8] was the genotype found most frequently. Following up children after discharge from hospital allowed the discovery of cases of nosocomial RVAGE which are missed in most other studies.