Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria, Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P, aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl, These results indicate that P, aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyl-tRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.