In an earlier report, it was shown that murine spleen cells cultured with concanavalin A (Con A) released into the culture supernatants helper and suppressor substances for antibody production. The present communication describes the production of rabbit antisera against culture supernates from Con A-activated spleen cells and their use in a plaque assay for mitogen-activated T cells. The plaque assay, utilizing SRBC to which Staphylococcal protein A had been coupled, the developing anti-supernatant antiserum and guinea pig complement, readily detected secreting T cells. The T-cell nature of the plaque-forming cells (PFC) was established principally by the following: (a) the majority of lymphocytes in the centers of plaques were Thy-1-positive by fluroescence; (b) spleen cells depleted of B cells by incubation in plastic dishes coated with rabbit anti-mouse Ig antibody gave greatly enriched PFC responses; (c) anti-Thy-1 and anti-Lyt-2.2 treatment of spleen cells almost completely depleted PFC; (d) T-cell mitogens (Con A and phytohemagglutinin) but not B-cell mitogens (lipopolysaccharides) induced PFC responses; (e) T cells maintained in culture for 10 d with Con A and T-cell growth factor yielded PFC. Kinetic and dose response studies showed that high doses of mitogen induced rapidly appearing T-PFC and the responses peaked at day 1--2 of culture. Lower doses of mitogen-induced PFC required longer periods of incubation for detection, indicating that cell activation and secretion may be different dose-dependent activities of mitogens. Another noteworthy finding was that the antiserum reacted with surface antigens of T-PFC, indicating that secreted products are expressed on the membranes of T cells, offering the possibility of isolating populations of cells with specific secretory potential. Although the precise nature of the T-cell products detected by the antiserum used in this assay are unresolved, 10% of the target-cell-adherent population from spleen cells of BALB/c mice sensitized to L929 cells formed plaques. This suggests that the antiserum has significant activity against the products of cytotoxic T cells, a finding which accords with the activity of anti-Lyt-2.2 serum against mitogen-induced T-PFC. The method clearly offers new possibilities for the analysis of T cells and their products and should provide an important approach to the clonal analysis of lymphokine production.