The ability of a number of metals and organic chemicals to induce metallothionein (MT) synthesis in primary cultures of rat hepatocytes was tested to determine whether MT induction in vivo results from a direct effect of the agent on the liver or as a result of an indirect, physiologic response to the agent. Hepatocytes were exposed to metals [zinc (Zn), cadmium (Cd), mercury (Hg), manganese (Mn), lead (Pb), cobalt (Co), nickel (Ni), and vanadium (V)] or organic compounds [ethanol, urethane, L-2-oxothiozolidine 4-carboxylate (L-OTCA), or dexamethasone] and were assayed for metallothionein by the Cd/hemoglobin radioassay. Cell viability was monitored by protein synthesis activity and cellular K+ concentration. Increases in MT concentrations were noted for Zn (22-fold), Hg (6.4-fold), Cd (4.8-fold), Co (2.4-fold), Ni (2.2-fold), and dexamethasone (4.5-fold). However, even at maximum tolerated concentrations, Mn, Pb, V, ethanol, urethane, and L-OTCA did not increase MT. The results indicate that Zn, Cd, Hg, Co, Ni and dexamethasone induce MT in vitro and thus are direct inducers of MT synthesis in hepatic tissue. In contrast, Mn, Pb, ethanol, urethane and L-OTCA, which did not increase the MT content of hepatocytes, apparently do so in vivo by an indirect mechanism.