- Culture supernatants of spleen cells from tumor‐bearing mice (TBM) have been found to enhance tumor growth, when injected together with tumor cells, and to augment the generation of antibody‐producing cells (APC), when injected into mice together with sheep erythrocytes (SRBC). In an attempt to characterize the cellular entity in the spleen‐cell population which is involved in release of the enhancing factor (EF), “B”, adult‐thymectomized, and nude mice were inoculated with 3LL tumor cells, and their spleen‐cell supernatants were tested. We found that spleen‐cell supernatants from the three different categories of 3LL tumor‐bearing mice were highly effective in augmenting anti‐SRBC APC production and in accelerating tumor growth. In a second line of experiments, T or B lymphocytes were eliminated from spleens of TBM before the cells were incubated in vitro. Culture supernatants of TBM spleen cells adherent to nylon wool colums, which were enriched mainly for B lymphocytes and macrophages, manifested higher EF activity than supernatants of eluted spleen cells which were enriched for T lymphocytes. Supernatants of TBM spleen cells, which were pretreated with anti‐Thy 1,2 serum in the presence of complement before culturing, were found to be highly effective in potentiation of APC generation and in enhancement of tumor growth. These results suggest that EF is not released by lymphoid cells that possess characteristics of mature T lymphocytes. EF was produced in culture by 3LL tumor cells; 90 min after injection of 3LL supernatant, the recipient spleen cells were capable of releasing EF in vitro. Trypsin treatment did not affect EF production by tumor cells but stopped its release from spleen cells of TBM. It therefore appears that EF is produced by the tumor cells and is then adsorbed onto spleen cells.