We previously described the interleukin 3 (IL-3)-dependent cell line, M1-A5, which has both natural cytotoxic (NC) and suppressor cell activities, the latter of which is mediated, in part, by the release of two cytokines which activate suppressor cells from unprimed lymphoid precursor cells. In this study we have compared the M1-A5 cell line with four other IL-3-dependent cell lines to determine whether these dual activities are universally associated with IL-3 dependence and to test the hypothesis that there is a direct relationship between the cytotoxic and the suppressive activities. The cell lines tested were a bone marrow derived Dexter culture derived line (FDC-P1), two Moloney leukemia virus induced leukemias (DA-1 and DA-3), and a mast cell line (PT18(A17]. All lines were dependent on IL-3 for survival but FDC-P1, DA-1, and DA-3 showed varying degrees of short-term proliferation in granulocyte-macrophage colony stimulating factor (GM-CSF). The cell lines all expressed asialo GM1 and Ly-5 surface markers but differed with respect to other markers. DA-1 expressed MAC-1, FDC-P1 and DA-3 expressed Thy-1, and PT18(A17) expressed receptors for the Fc portion of IgE. The cell lines varied greatly in their cytotoxic activity against WEHI-164. FDC-P1, DA-1, and PT18(A17) had low NC activity. DA-3 had consistently high activity, greater than that seen with M1-A5 cells. However, none of the cell lines secreted constitutively a suppressor cell inducing factor (SIF). In addition, it was demonstrated that recombinant murine TNF did not activate suppressor cells capable of inhibiting antibody synthesis and that anti-TNF did not block SIF activity, thus suggesting that TNF contamination of the M1-A5 derived SIF preparation is not responsible for the induction of suppressor cells. We conclude that suppressor cell inducing factors are not universally secreted by IL-3-dependent cell lines, that there is no correlation between NC and SIF activity, and that the dual activities of M1-A5 cells are not mediated by TNF.