Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV la and 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the la and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the la protein containing putative helicase and methyltransferase domains respectively. Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps. Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase. Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the la and 2a proteins. The greatest inhibition was shown by antibodies to a peptide containing the GDD motif. These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.