Macrophage interactions with extracellular matrix and other cells are important in phagocytosis, inflammation, and immunity. To learn more about the surface molecules involved in adhesion we compared the binding of murine macrophages and polymorphonuclear leukocytes (PMN) with artificial substrate in vitro. A distinctive type of adhesion of thioglycollate-elicited peritoneal macrophages (TPM) to bacteriologic plastic (BP) was defined, which was pronase-sensitive, Mg2+-dependent, and required cytoskeletal stabilization. A rat mAb designated 5C6 was isolated because it inhibited TPM attachment to BP, as well as mediating detachment of TPM adherent to that substratum. In addition, it inhibited the attachment of PMN to tissue culture plastic. This antiadhesive property of 5C6 mAb required intact IgG; the F(ab')2 fragment was partially effective and Fab was ineffective. 5C6 recognized the type 3 complement receptor, inhibiting rosetting of EAC3bi to TPM and immunoprecipitating a heterodimer of 160 and 95 kD that comigrated with the M1/70 immunoprecipitate. 5C6 recognized a pronase-stable epitope distinct from that of M1/70. Other mAbs, including M1/70 (CR3) and 2.4G2 (FcR), failed to have any antiadhesive effect in vitro. The inhibitory activity of 5C6 in short-term adhesion assays correlated with its inhibition of recruitment of myelomonocytic cells to a thioglycollate-elicited peritoneal exudate in vivo, after intravenous injection of mAb. 5C6 IgG inhibited recruitment of myelomonocytic cells by 84 +/- 3% at 1 d compared with saline-injected controls. The F(ab')2 fragment and a class-matched control IgG had little effect. Recruitment of TPM at 4 d was also efficiently inhibited by 5C6 IgG. 5C6 IgG was not cytotoxic, had no effect on marrow egress, did not cause increased phagocytic clearance of circulating neutrophils, and had no adverse effect on chemotaxis in vitro. We show that CR3 alone of the LFA-family is necessary for the recruitment of myelomonocytic cells to inflammatory stimuli such as thioglycollate broth. This strategy may be of general use in isolating reagents that inhibit the adhesive function of CR3 and provides a novel approach to antiinflammatory therapy.