Primary cultures of guinea pig tracheal epithelial cells maintained in an air/liquid interface system that maintains differentiated characteristics were grown to near confluence and exposed for 1 h to platelet-activating factor (PAF) on both apical and basal sides. PAF provoked release of high-molecular-weight mucin-like glycoproteins (MLG) from the cells, with maximal stimulation occurring at 10(-8) and 10(-9) M. The inactive form of PAF, lyso-PAF, was without effect. Indomethacin, the cyclooxygenase inhibitor, did not affect secretion stimulated by PAF, but nordihydroguiaretic acid (NDGA), a mixed cyclooxygenase and lipoxygenase inhibitor, attenuated secretion stimulated by PAF in a concentration-dependent manner. High performance liquid chromatography assay of the culture medium after addition of PAF revealed increased production of 15-, 12-, and 5-hydroxyeicosatetraenoic acids (15-, 12-, and 5-HETEs). The stimulatory effect of PAF on both mucin secretion and formation of HETEs was inhibited by the PAF receptor antagonists, CV-3988 and Ro 19 3704, with Ro 19 3704 acting at a concentration 10-fold lower than CV-3988 in inhibiting both effects. When added exogenously to the cell cultures, the combination of 5-, 12-, and 15-HETEs stimulated MLG release in a concentration-dependent manner. The results suggest that PAF stimulates release of MLG by guinea pig airway epithelium in vitro by a mechanism involving binding of PAF to receptors on epithelial cell surfaces, stimulation of lipoxygenase metabolism of arachidonic acid to HETEs within the epithelium, and stimulation of secretion by these epithelial-derived HETEs via an autocrine or paracrine mechanism.