In vitro exposure to human serum albumin (HSA) of splenic lymphocytes from mice sensitized for delayed-type hypersensitivity (DTH) against HSA resulted in the release of substances that could induce a footpad inflammatory reaction with a maximum of 6 hr after injection into normal mice. The substances were fractionated mainly in a molecular weight range of 30,000 to 70,000 daltons on Sephadex G-200. The ability of sensitized lymphocytes to produce the substances was dependent on T cells, was antigen specific, and correlated well with the ability of the lymphocytes to mediate DTH reactions. Moreover, the substances were produced efficiently by the DTH effector cell population generated in the in vitro culture system and also by the effector cell-enriched fractions on discontinuous bovine serum albumin gradients. These results suggest that the substances are produced by DTH-effector cells.