A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.