Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.