Since a “low-dose and long-term” administration of erythromycin (EM) was reported to be effective in patients with chronic respiratory diseases, including diffuse panbronchiolitis (DPB), the modulation of host defense responses by EM has attracted much attention. Despite considerable controversy, it was recently demonstrated that macrolides reduced neutrophil function. In this study, we investigated the effects of EM, a 14-membered ring macrolide, azithromycin (AZM), a 15-membered ring macrolide, and rokitamycin (RKM), a 16-membered ring macrolide, on neutrophil function in terms of active oxygen generation of neutrophils in the absence and presence of mononuclear cells in vitro. EM and AZM significantly suppressed active oxygen generation by neutrophils in the absence of mononuclear cells at low concentration (0.5 μg/ml. p<0.05). At the next step, to confirm that EM and AZM directly reduced active oxygen generation by neutrophils, we investigated whether mononuclear cells affected this effect of EM and AZM. In the presence of mononuclear cells pretreated with EM or AZM, both antibiotics suppressed active oxygen generation at concentrations ranging from 0.5 to 20 μg/ml. However, the inhibition rates induced by EM and AZM at low concentrations were not so different between the absence and the presence of mononuclear cells. These results indicated that EM and AZM have direct effects on the active oxygen generation by neutrophils and those effects that were not influenced by mononuclear cells. This inhibitory effect may be responsible for the therapeutic efficacy of these 14-membered and 15-membered ring macrolides in patients with DPB.