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Enterovirus (EV) infections commonly cause fever in infants younger than 90 days of age. The polymerase chain reaction (PCR) has improved our ability to diagnose EV infections.To evaluate the utility of blood and cerebrospinal fluid (CSF) specimens for the diagnosis of EV infections by PCR and to describe a large cohort of EV-infected infants.Febrile infants younger than 90 days of age evaluated for sepsis at Primary Children's Medical Center in Salt Lake City, UT, were enrolled in a prospective study designed to identify viral infections from December 1996 to June 2002. All patients had bacterial cultures of blood, urine and CSF. Testing for EV was performed by PCR and/or viral cultures. Patients who were positive for EV were identified for this study.Of 1779 febrile infants enrolled, 1061 had EV testing and 214 (20%) were EV-positive. EV infections were diagnosed by PCR of blood, CSF or both in 93% of infants. PCR testing was positive in blood in 57%, and blood was the only positive specimen for 22% of EV infected infants. PCR of CSF was positive in 74%. The mean age of infants with EV infection was 33 days, with 18% younger than 14 days and 5% younger than 7 days. Fifty percent of EV-positive infants had CSF pleocytosis. Of EV PCR-positive infants, 91% were admitted, and 2% required intensive care. Possible serious EV disease was diagnosed in <1%, and there were no deaths. Twelve infants (5.6%) had concomitant urinary tract infection, and 3 (1%) had bacteremia.EV infections are common in febrile infants younger than 90 days. Blood and CSF are equally likely to yield positive results by PCR, but the combination of both specimens improved the diagnostic yield.