In order to elucidate the mechanism of metallothionein (MT) induction by bacterial endotoxin during acute phase alteration, I investigated direct and indirect inducers of MT by measuring the induced uptake of [(35)S] cysteine into cultured cells. Although zinc or dexamethasone induce MT directly when added to the culture medium of human hepatic (Chang) cells, endotoxin added to the culture medium was found to be ineffective in inducing MT synthesis. Since MT was induced during acute phase alteration, I focussed on the role of macrophages. I found that the conditional medium from endotoxin-activated macrophages ("MΦ+LPS") induced MT synthesis in Chang cells, while the incubation medium of nonactivated macrophages did not. Primary induction by zinc, copper or cadmium in "MΦ+LPS" was denied, because the concentrations of these metals in "MΦ+LPS" were almost the same as in the control medium. To clarify the details of the process of MT induction, the kinetics of MT synthesis by "MΦ+LPS" in Chang cells were studied and compared with the kinetics of MT synthesis by zinc and dexamethasone in Chang cells. "MΦ+LPS" induces MT synthesis, proportionally to the concentration of "MΦ+LPS", whereas zinc and dexamethasone induce MT sigmoidally and biphasically, respectively. On the other hand, the time course of MT induction by "MΦ+LPS" is similar to that by zinc, but different from that by dexamethasone. I conclude that: 1. Macrophages activated by endotoxin release a new factor which induces MT synthesis in human hepatic cells. 2. The new factor is different from dexamethasone and zinc.