Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.