A hospital-acquired outbreak with febrile illness and/or rash occurred in our neonatal special care nursery (SCN) from September 1995 to September 1996. A total of 23 infants developed symptoms. We could not detect the etiological agents by routine virus isolation. In a retrospective study, however, enterovirus RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from four out of six cerebrospinal fluid (CSF) samples and from two of 12 sera. Thus six out of 16 patients from whom samples were obtained were diagnosed retrospectively as having enterovirus infection. Furthermore, we detected the enterovirus genome from four of 20 serum samples obtained from patients who had other clinical symptoms, and from infants hospitalized without noticeable clinical illness during the same periods. This outbreak was caused by two different enteroviruses, which we assumed were echovirus type 7 (Echo 7) and coxsackievirus B3 (Cox B3), because of the sequence results. We demonstrated the clinical advantage of the analysis of nucleotide sequencing as supportive evidence of transmission.