The YE1/48 antigen, defined by two rat monoclonal antibodies YE1/48 and YE1/32, is a disulfide-linked dimer of 45- to 50-kDa subunits expressed on murine T cells. It has previously been described as a T cell receptor alpha/beta-like molecule because of its similar m.w., dimeric structure, and isoelectric points comparable to those of the murine T cell receptor. We have now further characterized this antigen, directly compared it with the T cell receptor, and obtained internal amino acid sequences from the purified and trypsin-digested antigen. Endoglycosidase F treatment revealed the peptide cores of the antigen to be approximately 32 to 38kDa under reducing conditions and they possess at least three glycosylation side-chains. On diagonal gel analysis (nonreducing vs reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the YE1/48 antigen and the T cell antigen receptor alpha/beta immunoprecipitated from thymocytes are indistinguishable. However, the two molecules can be distinguished on EL-4 cells by sequential immunoprecipitation using the YE1/48 monoclonal antibody and a rabbit antiserum reactive with the murine T cell receptor. Furthermore, two MBL-2 variant clones, which differ in the level of YE1/48 antigen expression by more than 200-fold, express comparable level of the T cell receptor. Therefore, the YE1/48 antigen and the T cell antigen receptor alpha/beta seem to be different molecules. The YE1/48 antigen was purified from MBL-2(4.1) cells by affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin and the resultant peptides were separated by reverse phase high performance liquid chromatography. The amino acid sequences of several of the YE1/48 tryptic peptides were determined. Upon comparison with the protein sequences in the data base, no identical sequences were detected. These results demonstrated that the YE1/48 antigen is clearly different from the T cell receptor alpha-, beta-, or gamma- chain gene products, and it is a novel T cell antigen not previously described. However, the possibility of homology with other proteins remains undetermined because the tryptic peptides are too short to yield meaningful statistical comparison with the data base.