The ATCC66 strain of brome mosaic virus (BMV) (propagated at Kyoto University) contained two types of coat protein in its virion whereas the Russian strain of BMV has been known to contain a single coat protein; both strains have two initiation codons for coat protein in the same reading frame at the 5'-proximal end of the gene in RNA 4. Comparative studies on the nucleotide sequences of the ATCC66 and Russian strains of BMV demonstrated that in the ATCC66 strain, two adjacent adenine residues were absent from RNA 3 in the leader sequences of the coat protein gene just a few nucleotides 5' to the first initiation codon of the coat protein gene. Using biologically active cDNA clones of BMV RNA of the ATCC66 strain, we inserted two adjacent adenine residues into the cDNA of RNA 3 to obtain an RNA 3 transcript which has the same nucleotide sequence as the Russian strain in the non-coding leader sequence of the coat protein gene. Barley protoplasts inoculated with this RNA 3 transcript together with RNA 1 and 2 produced a single coat protein. To obtain further insight into the mechanism of translation of the BMV coat protein, we constructed several types of RNA 4 by changing the sequence surrounding the first AUG codon in the coat protein gene and analysed the in vitro translation products of the mutant RNA 4. The results confirmed that the absence of the two adjacent adenine residues was responsible for the production of two types of coat protein in the ATCC66 strain. The deletion of the two adjacent adenine residues in ATCC66 resulted in a base substitution of A with U three nucleotides 5' to the first AUG in the coat protein gene. The base substitution reduced translational activity from the first AUG codon and concomitantly increased translational activity from the second AUG codon from which a truncated coat protein was translated.