We have recently isolated a monoclonal antibody directed against Escherichia coli RNA polymerase that does not inhibit transcription. This antibody is a useful tool to immobilize this enzyme for transcription assays or protein-protein interaction studies. The epitope of this monoclonal antibody was precisely located by a combination of protein deletion and synthetic peptide scanning. The amino acids of the epitope were also determined. We conclude that this antibody binds an epitope shared by several bacterial species and therefore can be used to characterize or purify other related enzymes.