Macrolide antibiotics such as erythromycin (EM) and azithromycin (AZM) are beneficial in the treatment of mucus hypersecretion in inflammatory pulmonary diseases. Several indirect and direct mechanisms of action have been proposed. This study investigates the direct effect of macrolides on secretory function of isolated submucosal mucous gland cells (SMGCs). We hypothesize that macrolides inhibit the calcium influx necessary for evoked mucus secretion. To test this, we quantified mucin protein release using enzyme-linked immunosorbent assay, calcium-activated K(+) (K(Ca)), and calcium-activated Cl(-) (Cl(Ca)) currents. We measured nonselective cation current (NSCC) using whole-cell patch-clamp techniques; intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 Ca(2+) imaging. We found that both EM and AZM are agonists at muscarinic receptors. EM (10 µM) evoked a small but significant increase in mucin release but inhibited the mucin release induced by subsequent acetylcholine (ACh) treatment. Both EM and AZM (10 µM) evoked K(Ca) and Cl(Ca) whole-cell currents, which were blocked by atropine. EM and AZM also accelerated the decay of inositol trisphosphate-induced K(Ca) and Cl(Ca) currents without changing the peak current amplitudes. Likewise, internal application of AZM (10 µM) enhanced the decay rate of ACh-induced K(Ca) and Cl(Ca) currents. EM (1-10 µM) and AZM (0.1-10 µM) slowly (over 25-30 min) inhibited thapsigargin (TG)-induced Ca(2+) entry when applied during the plateau phase of Ca(2+) entry but blunted TG-induced Ca(2+) entry by 70% after a 5-min pretreatment before initiating calcium entry. EM blocked TG-induced NSCC. We conclude that macrolide antibiotics are partial agonists at muscarinic receptors but inhibit stimulated mucus release by inhibiting calcium entry in SMGCs.