Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M1, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.