Nucleotide sequences of the mammalian orthoreovirus (reovirus) type 1 Lang and type 2 Jones M3 gene segments were newly determined. The nucleotide sequence of the reovirus type 3 Dearing M3 segment also was determined to compare with a previously reported M3 sequence for that isolate. Comparisons showed Lang and Dearing M3 to be more closely related than either was to Jones M3, consistent with previous findings for other reovirus gene segments. The microNS protein sequences deduced from each M3 segment were shown to be related in a similar pattern as the respective nucleotide sequences and to contain several regions of greater or less than average variability among the three isolates. Identification of conserved methionine codons near the 5' ends of the Lang, Jones, and Dearing M3 plus strands lent support to the hypothesis that microNSC, a smaller protein also encoded by M3, arises by translation initiation from a downstream methionine codon within the same open reading frame as microNS. Other analyses of the deduced protein sequences indicated that regions within the carboxyl-terminal third of microNS and microNSC from each isolate have a propensity to form alpha-helical coiled coils, most likely coiled-coil dimers. The new sequences will augment further studies on microNS and microNSC structure and function.