UM4D4 is a recently defined antigen that is expressed on approximately 25% of peripheral blood T cells, but on the majority of T cells in inflammatory synovial fluid. Anti-UM4D4 activates peripheral blood T cells in the presence of accessory cells and/or phorbol ester. UM4D4 has been assigned to a new antigen cluster termed CDw60. The present study examined the ability of anti-UM4D4 to activate T cell clones derived from the synovial fluid of patients with rheumatoid arthritis. UM4D4 was expressed at varying levels on both lectin-generated and antigen-specific clones, including clones of CD4+, CD8+, and CD4-CD8- phenotypes. Anti-UM4D4 used in soluble form as a single stimulus was typically mitogenic for the CD4+ and some of the CD8+ clones, but not for the CD4-CD8- clones. Phorbol ester boosted the response to anti-UM4D4 in some clones, had no effect in others, and diminished the responses in some cases. In contrast to anti-UM4D4, anti-CD3 was generally not mitogenic in soluble form, although it was mitogenic when conjugated to beads. The data show that T cell clones derived from an inflammatory T cell infiltrate can be readily activated through the UM4D4/CDw60 antigen.