Mac-2, a 30-35-kDa galactose-binding protein, is synthesized at similar levels in murine peritoneal exudate macrophages whether recruited in response to an intraperitoneal pathogen Mycobacterium microti, to sterile inflammatory stimuli such as thioglycollate broth, or to concanavalin A. In elicited or activated macrophages up to 30% of Mac-2 is constitutively secreted, and secretion is stimulated markedly by calcium ionophore A23187. Only thioglycollate-elicited macrophages express cell surface Mac-2, and binding is mostly (> 80%) a result of affinity for cell surface carbohydrate structures. Mac-2 surface expression is markedly reduced upon further activation of thioglycollate-elicited macrophages with bacterial lipopolysaccharide in vitro. Polylactosamine structures are present on all macrophage populations examined as determined by binding of Lycopersicon esculentum lectin, whereas alpha-galactosyl residues detected by Griffonia simplicifolia isolectin B4 are expressed only on the thioglycollate-elicited macrophages, indicating that these residues are the major determinants responsible for Mac-2 surface expression. Chemical cross-linking experiments have identified binding of endogenous cell-surface Mac-2 to three glycoproteins of molecular masses of 92, 125, and 180 kDa containing alpha-galactosyl and polylactosamine structures on thioglycollate-elicited macrophages. The restricted cell surface distribution of Mac-2 on thioglycollate-elicited peritoneal macrophages, a population of recently recruited monocytes, suggests a role(s) in early events of macrophage infiltration and tissue fixation such as extravasion and cell-matrix interactions.