A pulmonary macrophage-monocyte-derived mucus secretagogue (MMS) oligopeptide has been previously reported to induce mucus secretion in an in vitro model system with human airway explants and secretory epithelial cells. To understand the possible role of macrophages in the regulation of secretion of mucus, our laboratory has used a series of human macrophage hybridomas that were generated by fusing an hypoxanthine guanine phosphoribosyl transferase-deficient promonocytic line, U937, with macrophages obtaining by maturing monocytes in Teflon bags. The cell lines were proven to be true hybridomas by acquisition of donor class I antigens, additional chromosomes, as well as macrophage specific (maximum velocity) not present on the U937 parent line. One clone, clone 63, produced large amounts of an oligopeptide with an approximate molecular weight of 2000, which was identified from culture supernatants by ultrafiltration, chromatography, isoelectric focusing, and Western blot. Processed clone 63 supernatant had biologic activity causing increased secretion of radiolabeled glycoconjugate in both cultured airways and secretory epithelial cells. Immunoblot analysis with a polyclonal rabbit antisera generated against MMS was positive, and Western blot analysis produced a band at approximately 2000 daltons, consistent with the previously described MMS. MMS secretion could be stimulated by zymosan and lipopolysaccharide and inhibited by both cycloheximide and erythromycin. Dexamethasone had a different effect, appearing to stimulate MMS production intracellularly but inhibiting its release once it was synthesized. The availability of cloned hybridomas allows for study of the regulation of mucus secretagogue production as well as purification of molecular species and provides a valuable tool for the study of mucus secretion.