Translational initiation of most eukaryotic mRNAs occurs when a preinitiation complex binds to the 5' cap, scans the mRNA, and selects a particular AUG codon as the initiation site. Selection of the correct initiation codon relies, in part, on its flanking residues; in mammalian cells, the core of the "Kozak" consensus is R-3CCAUGG+4 (R=purine; the A residue is designated position +1). The R-3 is considered the most important flanking residue, followed by G+4. Picornaviral mRNAs differ from most cellular mRNAs in several ways; they are uncapped, and they contain an internal ribosome entry site that allows the ribosome to bind near the initiation codon. The initiation codon of coxsackievirus B3 (CVB3) is flanked by both R-3 and G+4 (AAAATGG). Here, we report the construction of full-length CVB3 genomes that vary at these two positions, and we evaluate the effects of these variant sequences in vitro, in tissue culture cells, and in vivo. A virus with an A-->C transversion at position -3 replicates as well as wild-type CVB3, both in tissue culture and in vivo. This virus is highly pathogenic, and its sequence is stable throughout the course of an in vivo infection. Furthermore, the in vitro translation products from this RNA are very similar to the wild type. Thus, R-3-thought to be the most functionally important component of the Kozak consensus-appears to be dispensable in CVB3. In contrast, a G-to-C transversion at G+4 is lethal; RNAs carrying this mutation fail to generate infectious virus either in tissue culture or in vivo. However, in vitro analysis indicates that G+4 has only a marginal effect on translational initiation, especially if R-3 is present; instead, the G+4 is required mainly because the second triplet of the polyprotein open reading frame must encode glycine, without which infectious virus production cannot proceed. In summary, our data indicate that CVB3 remains viable, even in vivo, in the absence of R-3, and we propose that the most important factor contributing to the high frequency of G+4-not only in CVB but also in other eukaryotic mRNAs, and thus in the consensus motif itself-may be the constraint upon the second amino acid rather than the requirements for translational initiation.