A prior study examining differences in the activities of erythromycin and azithromycin on cellular functions in the Gram-negative pathogen, Haemophilus influenzae, revealed a marked difference in their inhibitory activities. The study revealed that protein synthesis and 50S ribosomal subunit assembly were equal targets for inhibition by azithromycin while erythromycin was a preferential inhibitor of translation. This contrast in inhibitory activities stimulated a comparative analysis of three additional antibiotics: clarithromycin, flurithromycin and roxithromycin. Each compound was tested over a concentration range for inhibitory effects on cellular processes. Clarithromycin was the most effective inhibitor of protein synthesis with an IC50 of 5.6 mug/mL, followed by flurithromycin at 6 mug/mL, and roxithromycin at 9 mug/mL. IC50 values for antibiotic effects on viable cell counts and growth rates were similar to those obtained for protein synthesis. Flurithromycin had the strongest effect on 50S ribosomal subunit formation with an IC50 of 8 mug/mL, followed by clarithromycin and roxithromycin, at 9.0 mug/mL and 12.5 mug/mL respectively. 30S ribosomal subunit formation in cells treated with flurithromycin and roxithromycin was also reduced to some extent. Pulse-and-chase labeling kinetics examining subunit assembly rates verified the slower synthesis rate of the subunits in the presence of each macrolide. The results are discussed in terms of structural differences of these macrolides and their differential inhibitory effects on both cellular targets.