BACKGROUND Enterovirus infections are common in neonates. Virus isolation is the only diagnostic method to confirm enterovirus serotype infections, however, is not always successful. OBJECTIVE A new approach for the diagnosis of enterovirus infections was performed, using the reference strain inferred from sequence analysis of PCR products. METHODS Virus isolation, enterovirus RT-PCR and sequence analysis were performed from clinical samples or stored sera from two neonates with fever and rash. Neutralizing test (NT) antibodies against prototype reference virus were measured in paired sera. RESULTS Virus isolation was negative in both patients but the enterovirus genome was amplified in the acute phase sera obtained from the two patients. From the results of sequence analysis of 109 nucleotides located in the 5'-noncoding of the conserved region of enteroviruses, a high homology to echovirus types 25 and 30 was found. More than a 4-fold increase in NT antibodies against reference viruses was demonstrated in the acute and convalescent phase sera. They were confirmed as echovirus type 25 and 30 infection, respectively. CONCLUSIONS These virological examinations are practical and useful for clinical settings for a diagnosis of enterovirus infections because of an insufficient positive rate in virus isolation.