Mechanisms of macrophage-mediated cytotoxicity against a tumor-cell line (L-929 cells) were analyzed. Culture supernatants were harvested from mouse peritoneal macrophages cultivated for 3 days in the absence or presence of the stimulating agents Escherichia coli endotoxin or zymosan. The supernatants from stimulated cultures were cytotoxic for the tumor cells, evaluated by measuring release of radio-activity during subsequent 4 days' culture of 14C-thymidine-labelled tumor cells in the supernatants. Cytotoxicity was verified by counting cells per culture. Corresponding results were obtained from co cultures of stimulated macrophages and tumor cells, in accordance with a previous study. Selective release of af lysosomal enzyme (beta-glucuronidase) was shown in the supernatants from endotoxin- or zymosan-stimulated cultures, while reduced levels of glucose were seen in all supernatants from macrophage cultures. Dialysis of supernatants against fresh medium reduced the toxic activity somewhat. Dialysis restored the glucose content to optimal levels, while the enzyme activity was unchanged. Heating of supernatants to 56 degrees C for 30 min reduced the cytotoxicity along with a reduction in enzyme activity; 70 degrees C for 30 min removed both cytotoxic activity completely. Heating had no effect on the glucose content of the supernatants. The present data indicate that macrophage-mediated tumor cytotoxicity may be performed through release of heat-labile soluble factor(s) which co-variate with the secretion of a lysosomal enzyme from stimulated macrophages.