Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N‐terminal methionine residue of ribosome‐synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high‐copy‐number plasmid and selection for utilization of the substrate analogue formyl‐leucyl‐methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl‐tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294‐4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10‐formyl‐tetrahydrofolate, the formyl donor in methionyl‐tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy.