The levels of viral proteins in infected cells are thought to be regulated by a variety of mechanisms. The initiation codons for the PB1 and NA proteins of A/WSN/33 (H1N1) influenza virus are in a suboptimal Kozak sequence for translation. To determine the significance of these suboptimal Kozak sequences, model vRNAs, whose coding regions were replaced with the reporter SEAP gene (for secreted alkaline phosphatase) and recombinant viruses with optimal Kozak sequences for PB1 and NA were constructed. Conversion of the upstream sequence of the PB1 and NA initiation codon to an optimal Kozak sequence was reflected in the level of reporter protein expression, but not the level of PB1 and NA protein expression. The recombinant viruses that had optimal Kozak sequences for PB1, NA, or both genes had similar replicative properties, both in cell culture and in mice, to those of the wild-type virus. These results suggest that expression of the PB1 and NA proteins is regulated by a mechanism other than that controlling the initiation of translation of these proteins.