A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.