The distribution of MEL-14+ lymphocytes was investigated by both fluorocytometric analysis and complement-dependent-cellular-cytotoxicity (CDCC) tests in which rabbit anti-rat Ig was added with complement at a secondary step. When CDCC was employed to detect MEL-14+ cells, almost half of the thymocytes were found to be MEL-14+ in various strains of mice. This high proportion of MEL-14+ cells stands in striking contrast to prior reports. Furthermore, when determined by fluorocytometric analysis, MEL-14+ cells were found to comprise more than 80% of the cells in the thymus. The MEL-14+ thymocytes comprised both immature subsets (CD4-8-, CD4+8+) and mature subsets (CD+8-, CD4-8+). MEL-14 brightly positive (MEL-14high) cells, however, were located mainly in mature T cell subpopulations within the thymus. The MEL-14high thymocytes appeared to be susceptible to the CDCC method. Most of MEL-14+ cells present in spleens and lymph nodes were shown to be included in the MEL-14high population. The MEL-14+ cells susceptible to treatment with MEL-14, rabbit anti-rat Ig plus complement in the spleen and lymph node were restricted to cells of the T-lineage. These data suggest that T cells may change from cells with low expression of the MEL-14 antigens at their surface to cells with high MEL-14 antigens in the process of differentiation. Furthermore, these findings indicate that MEL-14 molecules may be used as a surface marker to characterize an important T cell subpopulation.