The effects of erythromycin on the formation of ribosomal subunits were examined in wild-type Escherichia coli cells and in an RNase E mutant strain. Pulse-chase labelling kinetics revealed a reduced rate of 50S subunit formation in both strains compared with 30S synthesis, which was unaffected by the antibiotic. Growth of cells in the presence of [14C]-erythromycin showed drug binding to 50S particles and to a 50S subunit precursor sedimenting at about 30S in sucrose gradients. Antibiotic binding to the precursor correlated with the decline in 50S formation in both strains. Erythromycin binding to the precursor showed the same 1:1 stoichiometry as binding to the 50S particle. Gel electrophoresis of rRNA from antibiotic-treated organisms revealed the presence of both 23S and 5S rRNAs in the 30S region of sucrose gradients. Hybridization with a 23S rRNA-specific probe confirmed the presence of this species of rRNA in the precursor. Eighteen 50S ribosomal proteins were associated with the precursor particle. A model is presented to account for erythromycin inhibition of 50S formation.