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Vaccination against rotavirus is protective against severe disease. Surveillance of rotavirus infection in developing countries might direct vaccination policy more efficiently.We implemented WHO's generic protocols for hospital- and community-based surveillance of rotavirus gastroenteritis. From April 2001 to May 2002, and from January 2003 to June 2003, we conducted hospital surveillance for rotavirus infection at the only pediatric ward in the capital of Guinea-Bissau. Children less than 5 years of age admitted with diarrhea or developing diarrhea during hospitalization were enrolled in the study. Rotavirus infection was detected in the feces samples using an ELISA assay. Rectal swabs were also obtained and its use was validated against stool specimen.During the surveillance period, 161 cases of rotavirus infection were registered. During the season, rotavirus accounted for 35% of all hospitalized diarrhea cases. The rate of nosocomial disease was 1.6 per 1000 child-days (95% confidence interval [CI] = 1.02–2.51) with high rates for children aged 12 to 23 months of age (rate: 3.09; 95% CI = 1.47–6.48). Most of the rotavirus cases (93%) were in children less than 2 years of age and only 10 children aged less than 3 months were infected. Fever (risk ratio (RR) 1.56; 95% CI = 1.16–2.10) and vomiting (RR 1.38; 95% CI = 1.11–1.73) were more common in patients with rotavirus than in patients with nonrotavirus diarrhea. The case-fatality was 8%. Results from stool samples and rectal swabs were concordant in 96% of the pairs. Rectal swabs increased the detection of rotavirus cases by 6% and deaths by 33% over stool sample results.Rotavirus infections were confined to a 4-month period each year. It is an important cause of childhood diarrhea with high case-fatality ratio in Guinea-Bissau. The use of rectal swab appeared to increase the detection rate of rotavirus infection and the case-fatality rate. The high rate of nosocomial infections in hospitalized children emphasizes the need for prevention of disease.