Maize chlorotic mottle virus (MCMV) is a 30-nm icosahedral plant virus composed of a single 25-kDa capsid protein component and a 4.4-kb single-stranded, positive-sense genomic RNA. Northern blot hybridization analysis detected a single 3'-terminal 1.1-kb subgenomic RNA in infected plants. Virion RNA directs the synthesis of several polypeptides in a rabbit reticulocyte lysate in vitro translation system of which only the 25-kDa polypeptide is immunoprecipitated by MCMV capsid protein antiserum. The 1.1-kb subgenomic RNA is a highly efficient messenger RNA for capsid protein synthesis. Positive polarity in vitro transcripts from 3'-proximal MCMV cDNA clones direct the synthesis of the capsid protein in in vitro translation experiments. These data suggest that the MCMV capsid protein is expressed from a subgenomic RNA in vivo, and that the 25-kDa capsid protein is encoded by the 3'-proximal open reading frame in the MCMV genome.