We have examined the possible function(s) of the protein VP3 encoded by the rotavirus SA11 genomic segment 3. Viral-associated VP3 in double-shelled and single-shelled particles was shown to bind GTP covalently and reversibly. These properties are similar to the unique characteristics of eukaryotic and viral guanylyltransferases, suggesting that VP3 is associated with a capping enzyme activity. Previous studies have shown that intact viral particles are required for transcription, making it difficult to unequivocally identify the functions of individual proteins within such particles. Characterization of VP3 produced in the baculovirus expression system showed that the expressed VP3 covalently bound GTP. These studies suggest that VP3 alone is the guanylyltransferase. GTP binding also was seen in core virus-like particles and single-shelled virus-like particles that lacked viral nucleic acid and were assembled in insect cells.