Affinity-purified rabbit antibody to the human C3b receptor (CR1) cross-reacted with an antigen that was expressed on murine splenocytes, lymph node cells, thymocytes, peritoneal macrophages, erythrocytes, and L929 cells, as assessed by flow cytofluorographic analysis of indirectly stained cells. The cell surface antigen recognized by antihuman CR1 had a Mr of 60,000 to 65,000 on each cell type, and the cross-reactive epitope(s) was sensitive to reduction with dithiothreitol but not to oxidation with NaIO4. Analysis by affinity chromatography of murine splenic B lymphocytes identified two cell surface proteins capable of binding to guinea pig C3b (C3bgp). The larger polypeptide had a Mr of 210,000, was not present in L929 cells, and may represent the murine CR1. The smaller polypeptide of 65,000 Mr was also present on L929 cells and was shown to constitute the cross-reactive antigen. Adsorption of detergent lysates of L929 cells with C3bgp-Sepharose depleted by 44% the antigen recognized by anti-human CR1; the C3bgp-binding protein and the cross-reactive antigen exhibited similar patterns on two-dimensional gel electrophoresis; and the isolated C3b-binding protein could be immunoprecipitated with anti-human CR1. Thus, the murine cell surface protein, termed p65, that is antigenically cross-reactive with human CR1 shares a capacity for binding to C3b in its detergent-solubilized form but is distinct from murine CR1 in its lower Mr, wider cellular distribution, and inability to mediate the adherence of C3b-coated particles in its native, membrane-associated form.