A monoclonal antibody, 6E9 established from mice injected with dolphin peripheral blood lymphocytes (PBLs) was characterized. In addition to its reactivity against 89.4% of dolphin PBLs, 6E9 reacted with 33.1% of bovine PBLs of which 22% were CD5+, 11.1% were CD5-. 6E9 recognized a 34 kD protein on the surface of dolphin and bovine PBLs. Analysis of the protein's N-terminal amino acid sequence indicated that 6E9 recognizes bovine major histocompatibility complex (MHC) class II antigens. These results suggested that 6E9 recognized MHC class II antigens on bovine PBLs. As we have already produced an anti-dolphin MHC class I monoclonal antibody, analysis of immune system using these monoclonal antibodies will advance our understanding of the evolution of the mammalian immune system.