In bacteria, as well as in chloroplasts and mitochondria, the free amino group of the methionylated initiator tRNA(fMet) is specifically modified by the addition of a formyl group. The importance of this modification remains unclear. With the availability of pure Escherichia coli 10-formyltetrahydrofolate:L-methionyl-tRNA(fMet) N-formyltransferase, the enzyme catalyzing Met-tRNA(fMet) formylation, the corresponding fmt gene and its flanking regions were cloned and sequenced. The chromosomal fmt gene was disrupted, and strains modified in their formylation activity were constructed. A depletion of the cellular formylation activity was accompanied by a decrease in the growth rate of the bacteria. At 37 degrees C, in a rich medium, the absence of a functional fmt gene reduced the growth rate to 0.28 doubling per h, from 2.3 for the control strain. At 42 degrees C, the studied fmt mutant strain did not grow further.