Virions of tobacco mosaic virus (TMV) are composed of a single strand of RNA, encapsidated in about 2130 copies of a coat protein of MW 17,500. Asselin and Zaitlin [Virology 91, 173-181 (1978)] demonstrated that virion preparations also contained small amounts of a second protein of MW 26,500, which they termed "H protein." H protein, detectable to an average frequency of one per virion, was thought to be a protein of host origin. Subsequent studies [Collmer, Vogt, and Zaitlin, Virology 126, 429-448 (1983)] showed the H protein was comprised of a backbone of TMV coat protein, linked by a postulated isopeptide bond to a small protein that probably was of host origin. The host-derived moiety of H protein is shown here to be ubiquitin, most probably coupled to the coat protein at lysine 53. This finding is based on microsequencing of the H protein, and is substantiated by immunoblotting analysis with antibodies to human ubiquitin. Conjugated ubiquitin was detected in virions of all five strains of the virus tested. To our knowledge, this is the first report of a ubiquitinated viral structural protein.