Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5' end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [alpha-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.