In determining the terminal sequences of the genomic dsRNAs of rotavirus by 5'-rapid amplification of cDNA ends (5'-RACE), it was found that most of the viral cDNAs contained extra nucleotides at their 5' termini that had not been reported before on any rotavirus sequence. Although the extra nucleotides could be dA, dC, dG, or dT residues, the extra nucleotides on the cDNAs usually consisted of a single dT residue. Experiments performed with DNA/RNA duplexes indicated that reverse transcriptase has an associated terminal nucleotidyl transferase (TdT)-like activity, which can add nontemplated nucleotides to the 3' ends of DNA, and that reverse transcriptase was responsible for the presence of the extra nucleotides detected on the 5'-RACE cDNAs. The TdT-like activity of reverse transcription was specific for double-stranded substrates (i.e., DNA/DNA or DNA/RNA duplexes) and was active over a wide range of temperatures and enzyme concentrations. Both commercially available Moloney murine leukemia virus and avian myeloblastosis virus reverse transcriptases contained the TdT-like activity. This work implies that 5'-RACE and primer extension assays must be used carefully in determining the terminal sequences of nucleic acids because, under standard reaction conditions, reverse transcriptase can add nontemplated nucleotides to the 3' ends of cDNAs following template-directed synthesis.