This paper presents data on reactions of murine macrophages with a variety of lectins, with special focus on Griffonia simplicifolia I-B4 isolectin, the only lectin we tried that distinguishes stimulated macrophages from resident populations. Specificity of Griffonia simplicifolia I reaction with carbohydrate determinants at the cell surface is shown by (i) ability of alpha-galactosidase treatment of intact cells to abolish all lectin binding whereas beta-galactosidase has no effect on lectin binding, (ii) ability of methyl alpha-D-galactopyranoside to completely inhibit lectin binding with methyl alpha-D-galactopyranoside having no effect on lectin binding, (iii) ability of brief treatment of intact cells with trypsin to liberate a glycopeptide but reacts with G. simplicifolia I to form a precipitate that is dissolved by addition of methyl-alpha-D-galactopyranoside or alpha-galactosidase, (iv) ability of methyl alpha-D-galactopyranoside (but no other monosaccharide) to completely inhibit avid binding of macrophages to G. simplicifolia I lectin immobilized on an insoluble support, and (v) ability of immobilized lectin to separate macrophages into highly pure subpopulations of lectin-reactive and lectin-unreactive cells, as shown by examination of fluorescein-labeled lectin-treated cells with phase-contrast/fluorescence microscopy.