We have compared the regulation of the human metallothionein (MT)-IIA gene by the cytokines tumour necrosis factor-alpha (TNF) and interferon beta (IFN-beta) in human fibroblasts. Both TNF and IFN-beta induced MT-II mRNA rapidly, but stimulation by TNF was more sustained. The effects of TNF and IFN-beta were further distinguished by the action of the protein synthesis inhibitor cycloheximide, which reduced MT-II mRNA stimulation by TNF but enhanced IFN-beta-induced MT-II mRNA. These results suggested that TNF and IFN-beta activate MT-II gene expression by partially distinct mechanisms. Consistent with this notion, combined treatment with both cytokines resulted in more than an additive level of MT-II mRNA induction. TNF and IFN-beta also acted cooperatively in inducing MT-II mRNA in HeLa cells. A reporter construct containing positions -765/+80 of the MT-II promoter linked to the CAT reporter gene failed to respond to either TNF or IFN-beta in HeLa cells, despite the presence of a putative IFN-stimulated response element (ISRE) and an activator protein-1 (AP-1) binding site, suggesting that these elements are insufficient for the activation of the MT-II gene by these cytokines. Thus induction of MT-II expression differs from the genes whose activation by TNF can be induced via the AP-1 element alone, as well as those genes whose activation by IFN is mediated solely through the ISRE site.