In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid beta subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500-575. A strong RifR mutation lying outside the Rif region, which changed Val146 to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for RifR mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong RifR mutations were obtained when Val146 was mutated, and several weak RifR mutations were also isolated near position 146. The results define a new, N-terminal cluster of RifR mutations, in addition to the classical central Rif region.