A new lymphocyte cell surface alloantigen, provisionally designated Ly 9, is detected by flow microfluorometry(FMF), as an extra specificity in sera from anti-Lyt immunizations. Ly 9.2, one of the allelic specificities, commonly is detected as a unique immunofluorescence profile of thymocytes with anti-Lyt 3.1 sera. The alternative antigenic specificity, Ly 9.1, can be detected routinely in antisera prepared against Lyt 2.1 and Lyt 3.2. This lymphocyte alloantigen has both a unique strain and cell/tissue distribution. Quantitative measurement of Ly 9 immunofluorescence by FMF on heterozygote cells shows that Ly 9 antigen expression is co-dominant and reduced in levels relative to parental homozygote cells. Ly 9 is expressed on all thymocytes, peripheral lymphocytes, and on a subpopulation of cells in the bone marrow. This antigen is not expressed to any significant degree on erythrocytes, epidermal cells, sperm, or in suspensions of testis, brain, kidney, liver, or lung. FMF analysis and absorption typing reveals a quantitatively lower level of antigen expression on thymocytes compared with spleen or lymph node cells. Cytotoxic elimination experiments confirm that Ly 9 is expressed on at least 2 different T cell functional subsets and B cells.