In this study we investigated the influence of serum ultrafiltrate on the activities of azithromycin, other macrolides and unrelated antibiotics against Escherlchia coli. In the presence of serum ultrafiltrate the MIC of azithromycin was decreased by 10-fold. The activities of erythromycin and roxithromycin were also enhanced, although to a lesser extent The potentiation of activity was inhibited by divalent cations and by pre-treatment of the ultrafiltrate with trypsin. Potentiation of azithromycin activity was associated with a pH-independent, early increase in bactericidal activity and inhibition of bacterial metabolism. We postulate that low molecular weight proteinaceous components of normal human serum interact with azithromycin and other macrolides to alter the susceptibility of Gram-negative bacteria to the antibiotics, possibly through increased macrolide penetration across the bacterial cell membrane permeability barriers.