Human enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family. Other members of this family utilize an unusual mechanism of translation initiation whereby ribosomes are recruited internally to the viral RNA by an internal ribosome site (IRES) located in their 5′ noncoding regions (5′ NCR). Using dicistronic reporter constructs, we demonstrate that the 5′ NCRs of the 7423/MS/87 and BrCr strains of EV71 function as an IRES both in extracts and in cultured cells. Preincubation of translation extracts with purified coxsackievirus 2A protease cleaved elF4G, a component of the cap binding complex, resulting in a significant decrease in translation of capped mRNAs. In contrast, the translational efficiency of the EV71 IRES was enhanced under this condition, demonstrating that the EV71 IRES functions similar to other enterovirus IRES elements when components of the cap binding protein complex are cleaved. Finally, insertion of an upstream, out-of-frame start codon in the 5′ NCR of the EV71 genome inhibited IRES activity, suggesting that EV71 can be classified as a type I IRES, in which ribosomes first bind upstream of the initiation codon and then scan the mRNA until an appropriate downstream AUG start codon is encountered and protein synthesis commences. © 2003 Elsevier Inc. All rights reserved.