The present study was designed to examine the cellular requirements for the generation of the suppressor T cells induced in the presence of fetal calf serum in culture. When C57Bl/6 mouse spleen cells were cultured for 4-5 days, these precultured cells were shown in mixing experiments to suppress the generation of cytotoxic effector cells (CTL) against allogeneic P815 cells or the generation of anti-SRBC humoral response by freshly explanted C57Bl/6 spleen cells. Spleen cells cultured in the presence of silica (0.5 mg) for 4 days, did not develop suppressor activity. However, when silica was added 3 days after the start of the suppressor generation culture, the development of suppressor cells was only slightly affected, although the phagocytic activity of these spleen cells was still totally abolished. When plastic or G-10 Sephadex column nonadherent spleen cells were cultured alone for 4-5 days, these cells did not suppress the generation of CTL or anti-SRBC humoral response. When the nonadherent spleen cells were cultured with plastic adherent spleen cells, however, suppressor cells developed and the suppressor activity of these cells was dependent on the number of adherent spleen cells co-cultured with the non-adherent spleen cells. This activity of the adherent spleen cells was insensitive to treatment with anti-Thy 1.2 serum plus complement and to X-irradiation. Furthermore, adherent PEC could not substitute for adherent spleen cells, indicating a possible tissue specificity for the macrophages in the adherent cell fraction which can function in supporting and/or accelerating the differentiation of "immature" suppressor T cells. Finally, culture-induced suppressor T cells were sensitive to X-irradiation and their activity was refractory to IL2 (TCGF), whereas the activity of alloantigen-induced suppressor cells was sensitive to IL2.