Identification of a subpopulation of lymphocytes producing lymphokines was attempted by fractionating the lymph node cells from guinea pigs immune to DNP-BSA by velocity sedimentation at 1 x G. Each of six fractions obtained by this procedure was cultured with or without the presence of antigen, and the culture supernatants that were separated 24 hr later were assayed for various lymphokine activities. Most of the lymphokines, including migration inhibition factor, chemotactic factor for neutrophils, mitogenic factor, and lymphotoxin were generated by the first two fractions of lymphocytes, which represented the largest, most rapidly sedimenting cells. Although th procedure of cell separation does not depend on cell surface properties, the larger cells contained more cells with T cell surface markers and the smalller contained more cells with B cell surface markers. Proliferative response of those lymphocytes measured by 3H-thymidine uptake, however, has shown that the largest two subpopulations responded poorly either to specific antigens or to mitogens (PHA and LPS), and rather that the medium size cells responded most strongly to the both stimulants. These results indicated that the production of some lymphokines confined to certain subpopulations of lymphocytes which are large in size. Further, these cells are readily separable from the medium sized cells that respond strongly to antigenic and mitogenic stimuli with mitogenic responses.