Rheumatoid arthritis (RA) synovial fluid (SF) T cells express the activation markers CD69, HLA-DR and very late antigen (VLA)-1, but surprisingly few bear interleukin-2 receptors (CD25). This unusual activation state is commonly assumed to be due to stimulation by local antigen, yet T cells activated in vitro express activation antigens in the clearly defined sequence: CD69, CD25, HLA-DR and finally VLA-1. Two possible explanations for the activation state of SF cells are: first, they comprise several subpopulations each expressing different activation antigens or, second, activation markers are up-regulated by mechanisms other than antigen stimulation. To examine these hypotheses, double- and triple-color immunofluorescence techniques were applied to four T cell populations: normal peripheral blood T cells activated in vitro, RA SF T cells, T cells from an in vivo model of migration [tuberculin purified protein derivative (PPD)-induced skin blisters] and T cells co-cultured with endothelial cells (EC). The results confirmed that in vitro activated T cells expressed activation markers in the sequence described above, with significant CD25 expression and few cells co-expressing CD69 with HLA-DR or VLA-1. In contrast, almost half the SF T cells were CD69+HLA-DR+ but CD25-; a significant minority were CD69+VLA-1+. T cells from PPD-induced skin blisters were already HLA-DR+ and VLA-1+ at 24 h, although, in vitro, PPD-activated T cells up-regulated HLA-DR and VLA-1 only after 1 week, suggesting that pre-activated T cells were preferentially recruited into the blisters. Finally, T cells were found to up-regulate CD69 and, to a lesser extent, HLA-DR after adhering to EC in vitro. In summary, the paradoxical activation state of SF T cells cannot be explained solely by single or multiple rounds of activation in situ. At least two other mechanisms, the preferential recruitment of pre-activated T cells and the induction of HLA-DR and especially CD69 by endothelial contact during migration, may also play a role.