The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We have determined the nucleotide sequence of cDNA derived from the middle-size RNA segment. The coding sequences of three proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through the analysis of isolated proteins. In contrast to our results with the small phi 6 dsRNA segment, the open reading frames are not tightly clustered. The homologous terminal noncoding regions between the middle and small dsRNA segments are found to be more extensive than RNA sequencing had previously indicated.