The expression of T cell-associated antigens was analyzed on extrathymically differentiated T cells from athymic nude (rnu/rnu) rats. Two-color flow cytometry and monoclonal antibodies were used to compare the subset structure of the rnu/rnu T cell population with that of normal T cells. In adult nude rats CD4+ and CD8+ cells were found which co-expressed the OX19 antigen (CD5) clearly defining them as T cells. Double-positive (CD4+CD8+) cells were not detected in nylon wool-enriched rnu/rnu T cells. The ratio of CD4+ to CD8+ cells in nude rat lymph node cells did not significantly differ from normal T cells. However, the composition of the nude rat CD4+ and CD8+ subsets [as identified by the subset-dividing monoclonal antibodies OX22 and P4/16 (anti-RT6)] differed from that found in control rats. The most striking observation among the rnu/rnu CD4+ subset was an inversion in the ratio of OX22+ to OX22- cells (0.5) compared to normal rats (2.3). Only 61% of the nude rat CD4+ cells were found to be RT6+ compared to 85% of rnu/+ CD4+ cells. In the rnu/rnu CD8 subset the proportion of RT6 co-expressing cells was also markedly reduced (38% vs. 77% of the CD8+ subset). Furthermore, the nude rat CD8+ subset contained a substantial number of OX22- cells which were nearly absent in normal rats. The demonstration of cells bearing T cell markers in adult athymic rats further confirms the existence of an extrathymic pathway of T cell differentiation. The unusual T cell subset composition found in athymic rats, however, indicates that T cell subset generation and/or maintenance along this pathway differs from normal T cell development.