Pure cultures of three types of mononuclear phagocytes-mouse peritoneal macrophages, unstimulated or after thioglycollate stimulation, and human monocytes-synthesize and secrete large amounts of lysozyme in vitro. The macrophage lysozyme is indistinguishable from authentic lysozyme in its ability to lyse M. lysodeikticus, inhibition by specific antisera, a similar size of 14,000 and cationic charge. Lysozyme secretion in culture is characterized by a large net increase in total lysozyme, 4-20-fold in 3 h, 75-95% of which is in the medium, and its continued extracellular accumulation over at least 2 wk in culture. Lysozyme is the major (14)C-labeled protein secreted into the medium by both unstimulated and thioglycollate-stimulated macrophages and the 0.75-1 microg produced per 1 x 10(6) cells/day represents 0.5-2.5% of the total cell protein. Lysozyme is a cell-specific marker for mononuclear phagocytes and the PMN, which contains preformed enzyme, since it is absent in lymphoid cells and a variety of fibroblast and epithelioid cell lines. Lysozyme production is also a useful measure of mononuclear phagocyte cell number. The rate of lysozyme production and secretion is remarkably constant for all cell types under a variety of culture conditions. Production by the mouse macrophage increases threefold on the 2nd day in culture and then remains linear with time. Production is optimal at a relatively low serum concentration, but can be maintained, in the absence of serum, in lactalbumin hydrolysate or, at a reduced level in basal media. The production and secretion of lysozyme are independent of the production of macrophage acid hydrolases. Net increase and secretion of lysozyme occur under conditions where acid hydrolases like N-acetyl beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, and cathepsin D are neither accumulated nor secreted. Massive phagocytosis of latex particles has no effect on lysozyme production and secretion. Lysozyme production can be rapidly inhibited by treatment with cycloheximide (0.4 microg/ml) whereas inhibition of its production by colchicine (10(-6) M) occurs only after a lag period of more than 8 h, and is probably due to a secondary effect. These results show that mouse macrophages provide a simple in vitro system to measure lysozyme secretion and its control. These studies also indicate the possible importance of mononuclear phagocytes in the secretion of a variety of biologically active products and in the modification of their environment.