Poliovirus translation is initiated at AUG743, 154 nt downstream of a conserved heptanucleotide CUUAUGG at the 3' border of the internal ribosome entry site. AUG586 is part of this motif and is normally not an initiation codon, but was activated following alteration of its context from CUUAUGG to ACCAUGG. Initiation at AUG586 was efficient and yielded a 7.2-kDa polypeptide translated in an open reading frame that overlapped AUG743 by 38 nt, but the presence of this activated codon reduced initiation at AUG743 by only 50%. Growth of a mutant poliovirus W1-5NC-1 containing the CUU-->ACC substitutions was impaired and was not alleviated by a termination codon placed four triplets downstream of AUG586 in the virus W1-5NC-2. The virus W1-5NC-6 contained the substitution U584A and had a similar sp phenotype; the phenotype of W1-5NC-1 is thus probably due to substitution within the conserved CUUAUGG motif per se rather than to activation of AUG586. A sp mutant virus W1-5NC-3 was derived from W1-5NC-1 by deletion of nt 588-745, indicating that AUG586 could initiate translation in vivo. These observations indicate that although AUG586 can be activated by upstream substitutions, it is nevertheless readily bypassed by ribosomes in mRNAs containing wt downstream elements, resulting in initiation at AUG743.