T lymphocytes (E rosetting cells) isolated from the joint fluid of four patients with juvenile rheumatoid arthritis (JRA) were first analyzed for surface antigen expression. Approximately 15% of cells were CD25+ (interleukin, IL, 2 receptor positive), in addition, a remarkable proportion of cells expressed the CD2+3 phenotype. CD3+ cells outnumbered the sum of CD4+ and CD8+ cells as well as the cells reactive with the WT31 monoclonal antibody (which recognizes a framework determinant of the α/β T cell receptor). Purified T cells were cloned under culture conditions (1% phytohemagglutinin, PHA plus IL 2) which allow clonal expansion of most peripheral blood T lymphocytes. Under these conditions proliferating cells ranged from 25 to 65%; clones (derived from microcultures containing 0.5 or 0.25 cells/well) were tested for cytolytic activity against P815 cells (in the presence of PHA) or against the natural killer (NK)-sensitive K562 target cells. Fifty-four percent and 73% of clones obtained from the two patients with the polyarticular form of the disease displayed cytolytic activity in the lectin-dependent assay. Cytolytic clones were 22 and 29% in the two patients with single joint involvement. About half of all cytolytic clones displayed NK-like activity. Surface antigen analysis revealed that, in addition to conventional CD3+4+8 and CD3+48+, a noticeable fraction of clones (50/202) displayed unusual surface phenotypes. In particular, 33/50 coexpressed CD4 and CD8 antigens; 7/50 were CD2+348 and displayed NK-like activity; 10/50 expressed CD3 but lacked both CD4 and CD8 antigen and did not react with the WT31 monoclonal antibody. In order to allow selective growth of IL 2-responsive cells, T lymphocytes were also cloned directly in IL 2. As much as 57% of all clones thus obtained (48/84) displayed cytolytic activity. Moreover, about half expressed unusual surface phenotypes including CD2+348, CD3+4+8+ and CD3+48WT31. Given the accumulation at the site of the joint involvement of unusual T cells, most of which displayed cytolytic activity and were likely to represent cells activated in vivo (IL 2 responsive), one may speculate that these cells may be involved in the injury process.