The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.