To determine the translation initiation potential of the 5' proximal four AUGs of the polycistronic P/C mRNA of Sendai virus, we have developed an efficient system for oligonucleotide-directed site-specific mutagenesis utilizing a plasmid vector which contains the replication origin of the single-stranded DNA phage f1 and the phage SP6 promoter. Utilizing uridine containing single-stranded DNA templates of the plasmid vector carrying the P/C gene we introduced point mutations in all four of the putative initiator codons (AUGs) of the P/C gene. Based on the analysis of in vitro translation products from the SP6 polymerase-directed transcripts of the mutants, the first and second AUG codons were assigned to the P and C proteins, respectively. Proteins detected in vitro, Y1 and Y2, initiated from the third and fourth AUG codons, respectively, and originated within the C reading frame. Since the C' protein is encoded in the same reading frame as the C protein but did not initiate from any of the 5' proximal AUGs, the synthesis of the C' protein may start from a non-AUG initiator codon in the P/C gene.