We have identified mutations in three different chromosomal genes of Escherichia coli K12 which reduce sensitivity to microcin B17. Mutations in ompF and ompR genes affected production of an outer membrane porin protein, OmpF, and resulted in reduced sensitivity to a number of other agents (colicins, bacteriophages) besides microcin B17. The third class of mutants were specifically and highly resistant to microcin B17. The mutations in these strains were mapped to a gene (sbmA), located at 8.7 min on the E. coli K12 chromosome, which is closely linked to phoA. The wild-type sbmA allele was cloned into multiple copy number plasmids, and its location within the cloned DNA fragment was further defined by mutagenesis with MiniMudII1681. These insertion mutations resulted in in-frame fusions between the sbmA and lacZ genes, thereby allowing us to determine the direction of sbmA gene transcription. Plasmids carrying these gene fusions produced low levels of beta-galactosidase, indicating that the sbmA gene is poorly expressed. We have been unable to identify the sbmA gene product, but indirect evidence indicates that it might be an envelope protein involved in microcin uptake.