It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.