A cDNA clone encoding the nonstructural, 30-kDa protein of the common (U1) strain of tobacco mosaic virus (TMV) was isolated and characterized. cDNA clones representing the intact gene as well as deletions from the 5' end of the gene were subcloned into SP6 vectors. Capped RNAs produced by in vitro transcription reactions were translated in a wheat germ cell-free system. The resultant proteins were compared to proteins obtained from the in vitro translation of intermediate length (I2) rods of TMV. Transcripts of the cDNA clones encoded polypeptides of 30, 28, or 18 kDa that were immunoprecipitated by antibody prepared against a synthetic peptide representing the carboxy terminus of the 30-kDa protein. cDNA clones containing the intact 30-kDa sequence coded for 30-kDa polypeptides while clones lacking the 30-kDa initiation codon produced 28-kDa polypeptides. Surprisingly, translation of a transcript from a cDNA clone containing the 30-kDa gene plus 390 nucleotides 5' of the initiator AUG yielded a polypeptide with an approximate molecular mass of 18 kDa. The results indicate that an intact and functional 30-kDa protein gene has been cloned. The significance of these results, with respect to determining the function of the 30-kDa protein, is discussed.