By immunizing mouse lymphoma cell line tMK-2U derived from intermediate TCR cells of BALB/c nude mouse, U5A2-13 monoclonal antibody (mAb, a rat IgG2a) was established. U5A2-13 antigen (Ag) was expressed on around 65% of TCRint cells in the liver of the various mouse strains including both NK1.1- and NK1.1+ mouse strains, while NK1.1 Ag was expressed only in NK1.1+ C57BL/6 mouse strain. Among CD3+ cells, 26.3% cells co-expressed U5A2-13 Ag and NK1.1+ Ag, while small proportions of the CD3+ cells were U5A2-13+NK1.1- (9.2%) or U5A2-13-NK1.1+ (4.4%). Among NK1.1+ cells, 54.9% cells co-expressed CD3 and U5A2-13 Ag, while some proportions of the cells were U5A2-13+CD3- (19.4%) or U5A2-13-CD3+ (9.8%). It was found that approximately 85% of NK1.1+CD3+ cells coexpressed U5A2-13 Ag. U5A2-13 Ag with low fluorescence intensity was also expressed on 55% of NK1.1+CD3-NK cells. U5A2-13 Ag immunoprecipitated from tMK-2U cells consisted of three proteins, which were 65 kDa, 33 kDa and 32 kDa under both reducing and non-reducing conditions and these were apparently different from NK 1.1 Ag. These results indicated that U5A2-13 mAb was able to define a similar population to NK1.1+CD3+T cells and to 55% of NK1.1+CD3-NK cells in various strains, through recognizing a different molecule from NK1.1 Ag.