L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated ‘tentacles’ that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance.