The OX2 membrane glycoprotein contains two immunoglobulin superfamily (IgSF) domains and seems likely to interact with other cell surface proteins. A soluble chimeric protein with the two IgSF domains of OX2 engineered onto domains 3 + 4 of rat CD4 antigen was expressed. To detect possible weak interactions, the chimeric protein was coupled to fluorescent covaspheres to provide a highly avid display of OX2. The OX2 covaspheres bound macrophages but not other cell types. The specificity of the interaction was demonstrated by blocking with Fab fragments of the OX2 monoclonal antibody (mAb). A new mAb, MRC OX88, was raised against macrophages which also blocked the interaction and presumably recognizes the ligand. The epitope for the MRC OX2 mAb and a site for ligand binding were mapped to domain 1 by site-directed mutagenesis. The OX2 antigen is present on thymocytes, some lymphocytes, neurons and endothelial cells; thus, it has the potential to mediate interactions between these cell types and macrophages.