Mechanisms of regulation of mouse metallothionein (MT)-I gene expression in response to bacterial endotoxin-lipopolysaccharide (LPS) were examined. Northern blot analysis of hepatic MT-I mRNA in interleukin (IL)-6 or tumour necrosis factor (TNF)-receptor type I knock-out mice demonstrated that IL-6, not TNF-α, is of central importance in mediating hepatic MT-I gene expression in vivo after LPS injection. In vivo genomic footprinting of the MT-I promoter demonstrated a rapid increase, after LPS injection, in the protection of several guanine residues in the -250 to -300 bp region of the MT-I promoter. The protected bases were within sequences which resemble binding sites for the signal transducers and activators of transcription (STAT) transcription factor family. Electrophoretic mobility-shift assays using oligonucleotides from footprinted MT-I promoter regions showed that injection of LPS resulted in a rapid increase in the specific, high-affinity, in vitro binding of STAT1 and STAT3 to a binding site at -297 bp (TTCTCGTAA). Western blotting of hepatic nuclear proteins showed that the time-course for changes of total nuclear STAT1 and STAT3 after LPS injection paralleled the increased complex formation in vitro using this oligonucleotide, and binding was specifically competed for by a functional STAT-binding site from the rat α2-macroglobulin promoter. Furthermore, the MT-I promoter -297 bp STAT-binding site conferred IL-6 responsiveness in the context of a minimal promoter in transient transfection assays using HepG2 cells. This study suggests that the effects of LPS on hepatic MT-I gene expression are mediated by IL-6 and involve the activation of STAT-binding to the proximal promoter.