A simple and reproducible microassay for the quantitation of macrophage mediated cytotoxicity is described. The method is based on the measurement of absorbance at 630 nm of residual Giemsa stained target cells and effector macrophages using an automated densitometer. Applying this novel method, it was possible to demonstrate time dependent growth characteristics of C3H/MCA and BHK/Py target cell lines. Using C3HeB/FeJ or C3H/HeJ murine effector macrophages and syngeneic transformed fibroblast target cells (C3H/MCA or 3T12), the method was further applied to demonstrate: (1) dose related activation of macrophages by lipopolysaccharide (LPS) and by macrophage activating factor (MAF); (2) synergistic augmentation of MAF-mediated macrophage cytotoxicity by LPS; (3) unresponsiveness of C3H/HeJ macrophages to LPS; and (4) increased cytotoxicity with increasing effector: target cell ratios. Guinea pig peritoneal macrophages were also shown to produce enhanced LPS or MAF-mediated cytotoxicity for C3H/MCA or BHK/Py target cells. The novel method was shown to compare favorably with results obtained by cytotoxic release of [3H]thymidine from prelabeled target cells. The advantages of the method are: (1) the elimination of the need for radioactive materials; (2) the ability to perform quantitation directly in microtiter plates; (3) the relative ease and rapidity in which experiments may be performed and quantitated; (4) its sensitivity and reproducibility; and (5) the ability to simultaneously quantitate and observe the biological events either microscopically or macroscopically.