An antiserum raised against a purified fraction of guinea pig lymphokines exhibits marked in vivo properties; it suppresses delayed hypersensitivity reactions and binds migration inhibition factor (MIF) from supernatants of activated lymphocyte cultures in vitro. This serum was analyzed for specificity using a radioactive double labeling technique which distinguishes products synthesized either de novo or in increased amounts by stimulated lymphocytes. Combined supernatants of stimulated (concanavalin A or PPD) and unstimulated cultures labeled with either tritiated or [14C]leucine, respectively, were fractionated on Sephadex G-75. Pooled fractions were precipitated using the antibody sandwich technique, and solubilized precipitates were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using this procedure the anti-lymphokine serum recognized primarily three newly sythesized lymphocyte activation products with molecular weights of approximately 60 000, 45 000 and 30 000 daltons. By contrast, these molecules could not be detected in radiolabeled lymph node cells which had been solubilized with NP-40 detergnt. The isoelectric point of all three molecules was found to be 5.2 +/- 0.3. The previously determined characteristics (mol.wt. 45 000, pI 5.2) of MIF and the property of the anti-lymphokine serum to absorb MIF activity suggest an identity between MIF and one of the molecules. In view of the previously described in vivo effects of the anti-lymphokine serum it is concluded that at least one of these three molecules plays an important role in the early events of delayed-type hypersensitive reactions in vivo.