Nine structurally similar macrolide antibiotics were tested at a concentration of 0.5 mu g/ml for their relative inhibitory effects on ribosome functions in Staphylococcus aureus cells. Eight of the compounds examined inhibited protein synthesis at this concentration. Seven of the nine compounds were also effective in blocking formation of the 50S ribosomal subunit. Roxithromycin and 14-hydroxy clarithromycin inhibited protein synthesis to a,greater extent than they affected 50S subunit formation. Conversely, the compound 11,12-carbonate-3 deoxy-clarithromycin affected 50S assembly mow than translation. Only clarithromycin had any effect on 30S ribosomal subunit assembly. The decline in growth rate and cell number was proportional to the effect on ribosome formation or function by each compound. These inhibitory activities can be related to structural differences between these macrolide antibiotics.