Azithromycin is an important antibiotic for the treatment of several different Gram-positive and Gram-negative bacterial infections. Erythromycin and clarithromycin are less useful antibiotics against Gram-negative infections. This difference in inhibitory activity was explored by comparing the effects of azithromycin and erythromycin on cellular functions in Haemophilus influenzae cells. Effects of both antibiotics on translation, cell viability, and growth rates have been measured. An IC50 of 0.4 mug/ml was found for the effects of azithromycin on each of these processes. For erythromycin, an IC50 of 1.5 mug/ml was observed, indicating a fourfold lower sensitivity of the organisms to this compound. The features of a second target for macrolide antibiotic inhibition in H. influenzae cells have also been examined. Inhibition of the synthesis of the large 50S ribosomal subunit was measured. Subunit formation was prevented in a concentration dependent fashion, with azithromycin showing a ninefold greater effect on this process compared with erythromycin. Synthesis of the 30S ribosomal subunit was not effected. Pulse and chase labeling kinetics confirmed the slower synthesis rate of the 50S particle in the presence of each antibiotic. The results are discussed in terms of the stronger effect of azithromycin on ribosome biosynthesis in this organism.