2B4 (CD244) is a 66-kDa CD2 family protein expressed on natural killer (NK) cells. Mouse NK cells express two isoforms of 2B4, termed 2B4L and 2B4S, whose molecular masses are 42 kDa and 36 kDa, respectively. In this study, we biochemically characterize the 2B4 antigen that was newly found on mouse bone marrow-derived mast cells (BMMC). Anti-2B4 mAb immunoprecipitated glycoproteins with a molecular mass of 60 kDa from BMMC. Removal of N-linked sugars from the antigen by N-glycosidase F treatment yielded two protein backbones of 35 kDa and 25 kDa, indicating that BMMC express the 2B4S isoform, but not 2B4L. Nucleotide sequence analyses confirmed that BMMC transcribe 2B4S mRNA. The preferential expression of the 2B4S isoform and the detection of an additional 25-kDa glycoprotein on BMMC indicate that differences in the structure of 2B4 antigen exist between BMMC and NK cells.