Activated macrophages have the capacity of selectively injure neoplastic cells. Morphological observations by others suggest that the final effectors of macrophage-mediated tumor cytotoxicity are lysosomes of activated macrophage origin which are translocated directly into susceptible target cells. The purpose of this study was to quantitatively determine if elevated levels of specific lysosomal activity are present in tumor cells exposed to activated macrophages in vitro. Effector macrophages were obtained from the peritoneal cavities of A/BiF/F50+ and C3H/HeN(MTV-) glucan-treated mice in which the mammary tumor virus is negative. Target cells were tumorigenic and nontumorigenic fibroblast cell lines derived from the same two strains. Macrophage-dependent target cell cytotoxicity was quantitated using [3H]thymidine incorporation inhibition and 51Cr postlabeling assays. Tumor lysosome activity was quantitated using newly developed microspectrophotometric assays for the lysosomal hydrolase acid phosphatase and for the lysosomotropic probe acridine orange. The results demonstrate that the specific lysosomal activity of tumor target cells correlates directly with the degree of tumor cytotoxicity generated by effector macrophages. Interestingly, this macrophage-induced elevation of tumor lysosome activity does not appear to represent the acquisition of macrophage-derived organelles; rather, it appears to represent an increase in the number or size of intact, endogenous tumor lysosomes due to macrophage-dependent reduction of tumor cell density. This finding suggest that clinically proven tumor growth-reducing regimens such as host macrophage activation may be useful adjuncts to cancer therapies designed to selectively promote and labilize tumor cell lysosomes.