Objective . To determine the presence of bacterial and viral infectious agents in children with fever during anticancer chemotherapy treatment. Design . Analysis of data obtained in a prospective cohort study. Setting . The paediatric oncology unit at Tygerberg Children’s Hospital, Stellenbosch University, Parow, Western Cape. Subjects . All patients up to the age of 15 years who developed fever secondary to anticancer chemotherapy from 9 February 2000 to 9 April 2001. Outcome measures . Viruses were isolated or antigens detected on venous blood samples, nasopharyngeal aspirates (NPAs), throat swabs, urine and faeces, where possible. Blood for aerobic and anaerobic culture was obtained from an indwelling intravenous catheter and/or a peripheral vein. Results . Thirty-four patients were analysed for a total of 102 febrile episodes. Evidence of a viral and bacterial infection was found in 31 (30%) and 24 (24%) episodes, respectively. Within these, a combined viral and bacterial infection was demonstrated in 6 (6%) episodes. A total of 35 viral isolates were identified in 31 febrile episodes: herpes simplex virus 1 (HSV-1) (N=14), HSV-2 (N=2), cytomegalovirus (CMV) (N=10), rotavirus (N=5), adenovirus (N=2), para-influenza type 3 (N=1) and hepatitis B (N=1). The blood culture was positive in 24 febrile episodes. The absolute neutrophil count (ANC) on admission was below 0.5 x 109/l in 57 (56%) episodes and thus considered neutropenic. Infectious agents were more frequently identified in neutropenic (54%) than in non-neutropenic (40%) episodes and were more likely to be of bacterial (30%) than viral (15.5%) origin. However, this difference was not significant. Conclusions . In addition to bacterial infections, viruses are clearly an important cause of fever in children receiving anticancer chemotherapy. Diagnostic tests for viral infections should be used more frequently, and could be of considerable value in evaluating fever and establishing appropriate therapy in these patients.