The 5'-terminal nucleotide sequences of human reovirus double-stranded RNA were determined after labeling the RNA with [(32)P]phosphate by polynucleotide kinase. The 5' terminal were labeled to only a limited extent prior to sequential oxidation, beta-elimination, and phosphomonoesterase treatment, indicating that the terminal phosphates were in a modified, blocked configuration. Each genome segment, after removing the blocking group, contained the same two 5'-terminal sequences: GpApUp in one chain and G(*)pCp in the other. G(*)p is a derivative of guanylic acid, probably 2'-O-methyl-Gp, which renders the 5'-terminal sequence resistant to hydrolysis by alkali. The results indicate that the transcription of reovirus double-stranded RNA strats from the 3' end complementary to the G(*)pCp-terminal, resulting in the synthesis of single-stranded mRNA carrying the same 5' sequence as the G(*)pCp-chain. The presence of a modified nucleotide at the 5' terminus of the strand complementary to the mRNA template is a feature common to another double-stranded RNA virus, cytoplasmic polyhedrosis virus.