The appearance of lymphotoxin in the culture fluid of phytohemagglutinin (PHA)-stimulated mouse spleen cells required two cell fractions that were separated by adherence to plastic. Upon stimulation with PHA, neither cell fraction alone produced significant amounts of lymphotoxin; however, when the cell fractions were combined and then stimulated with PHA, full activity was produced. Cytotoxic activity was not fully restored by combining PHA-stimulated cultured fluids from adherent and nonadherent cell fractions. This indicated that the cytotoxic activity was not the result of two factors, one produced by each cell fraction, that acted on the target cells, but rather, two cells interacted to produce lymphotoxin. Treatment of the unfractionated spleen cells with monoclonal anti-Thy1.2 and complement before PHA stimulation greatly reduced the production of lymphotoxin and indicated that at least one of the cells was a T cell. Lymphotoxin production was partially restored by the addition of nonadherent cells to the anti-Thy1.2-treated cells, suggesting that the T cell was nonadherent. Treatment of unfractionated cells with either silica or carrageenan had no effect on the subsequent production of lymphotoxin by PHA, suggesting that the adherent cell was not actively phagocytic.