The nucleotide sequence of the M2 gene segment of the mammalian reovirus prototype strain, type 3 Dearing, was determined from a cloned full-length cDNA copy of the viral double-stranded RNA segment. The gene comprises 2203 nucleotides and has a single long open reading frame that spans bases 30 through 2154 and encodes the 708 amino acid outer capsid protein mu 1. Aminoterminal sequence analysis of mu 1C, the proteolytically cleaved form of mu 1 that is found in purified reovirions, has identified the site of mu 1 to mu 1C cleavage between residues 42 and 43 in the mu 1 sequence. Aminoterminal sequence analysis of delta, the proteolytically cleaved product of mu 1C that is found in chymotrypsin-generated intermediate subviral particles, has indicated that the mu 1C to delta cleavage occurs near the carboxyterminus of mu 1C. Lastly, stoichiometric determinations using new sequence information have suggested that approximately equimolar amounts of mu 1C and the other major outer capsid component sigma 3 are present in virions. The data presented in this study should be useful for understanding the molecular basis of the functions of the mu 1 protein in reovirus entry into cells and in pathogenesis in the host animal.