The aim of this study was the detection of a 114 base pairs amplicon in 5' non-translated region of enterovirus genome in stool specimens of patients with acute flaccid paralysis (AFP) which were negative on cell culture. One hundred and twenty stool specimens were collected from AFP cases and tested with cell culture (RD, L20B and Hep2 cell lines). RT-PCR was carried out for the specimens with negative cell culture result. A 10% raise in enterovirus detection was observed with RT-PCR. This increased sensitivity can improve the detection of enterovirus serotypes which grow poorly in cell culture, and can thus alter significantly the medical care of patients with acute flaccid paralysis.