Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102–112) established that TruB, a pseudouridine (Ψ) synthase from Escherichia coli, was able to make Ψ55 in tRNA transcripts but not in transcripts of full-length or fragmented 16S or 23S ribosomal RNAs. By deletion of the truB gene, we now show that TruB is the only protein in E. coli able to make Ψ55 in vivo. Lack of TruB and Ψ55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type. The negative selection did not appear to be acting at either the exponential or stationary phase. Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 °C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to ∼0.2% of its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB. Direct analysis showed that this mutant did not make Ψ55 under the conditions of the competition experiment. Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of Ψ55.