Rotavirus multiplication was inhibited by neomycin B by affecting the synthesis of the viral genome. Genome replication involves a two-step mechanism, one is plus-strand synthesis or transcription, and the other is minus-strand synthesis or replication. The results indicate that both activities are inhibited by aminoglycoside. The effect on the minus-strand was determined by an in vitro assay using a template-dependant open core preparation. The effect on transcription was explored using transcriptionally active virus particles. In the case of transcription, the inhibitory effect of neomycin B was studied in both initiation and the elongation of the mRNA. Initiation was defined by the synthesis of short transcripts of less than 25 nucleotides in length and elongation as an extension of those molecules into full-length transcripts. The inhibitory effect of neomycin was also mimicked by other aminoglycosides such as lividomycin, paromomycin, and tobramycin. The results may be explained based on the ability of the drug to interact with the stem and loop regions, which, in the case of rotavirus, have been identified at the end of the templates required for both transcription and minus-strand synthesis.