The effects of several nucleoside and nucleoside triphosphate analogues were studied on rotavirus replication and transcription. Nucleoside triphosphate analogues modified at sugar residues were capable of inhibiting in vitro transcription, including adenosine-9-beta-D-arabinofuranoside 5'-triphosphate, 3'-deoxyadenosine 5'-triphosphate, adenosine 5'-triphosphate 2',3'-dialdehyde, guanosine 5'-triphosphate 2',3'-dialdehyde, and cytosine-9-beta-D-arabinofuranoside 5'-triphosphate. Two dialdehyde derivatives, adenosine 5'-triphosphate 2',3' dialdehyde and guanosine 5'-triphosphate 2',3'-dialdehyde, were irreversible inhibitors, forming a stable complex with the viral polypeptide VP3. The effect of the corresponding nucleosides of the inhibitory analogues was studied in SA-11 rotavirus-infected MA-104 cells. Adenosine-9-beta-D-arabinofuranoside and 3'-deoxyadenosine were effective inhibitors of RNA synthesis, an effect that could be due to their inhibition of viral transcription.