The resistance of Escherichia coli against the antibiotic rifampicin is caused by mutations in the rpoB gene which alter the structure of the beta subunit of the DNA-dependent RNA polymerase. By insertion-mutagenesis with bacteriophage Mu rifampicin-resistant mutants that were believed to have a sensitive RNA polymerase had been generated. For closer analysis of this putative rpoB-independent mechanism of resistance we cloned and sequenced the insertion sites from two of the Mu lysogens. One of them showed > 95% sequence similarity with the phn locus of E. coli B, and was mapped between nt 3824 and 3825 within the phnB gene. This positions the phn locus between 4397 and 4410 kb of the Kohara map of E. coli K-12. Since both analysed insertion sites differ in respect to each other and to the findings of previous work we determined the rifampicin sensitivity of the RNA polymerase of the Mu lysogens with partially purified enzyme. For all mutants the IC50 as a measure for sensitivity was significantly higher than for the parent strain. Sequence analysis of part of the rpoB gene (nt 1519 to 1716) revealed single point mutations in the investigated Mu lysogens. The rpoB mutation is necessary and sufficient for the observed resistance, while the prophage alone does not evoke resistance and has no synergistic effect together with the rpoB mutation. Our work supports the assumption that rifampicin like other hydrophobic molecules enters the cell via simple diffusion through the outer membrane with LPS being the main barrier.(ABSTRACT TRUNCATED AT 250 WORDS)