Although the existence of repressor protein(s) involved in the regulation of highly inducible metallothionein-I (MT-I) gene expression has been postulated, none has been identified to date. We considered nuclear factor I (NFL) protein as a potential repressor, as three half-sites for NFI binding are present on MT-I promoter and NFI is known to downregulate several cellular gene promoters. Overexpression of all four isoforms of mouse NFI protein (NFI-A, -B, -C, and -X) suppressed both constitutive and heavy metal-induced activation of the MT-I promoter in HepG2 cells. However, unlike other target genes of NFI, direct interaction of NFI with MT-I promoter is not necessary to mediate its repression. Point mutation of the NFI binding sites within the MT-I promoter that abrogates NFI binding in vitro could not alleviate the repression. Similarly, NFI proteins also repress activity of minimal MT-I promoter deficient in the NFI binding sites. Further, an NFI-C deletion mutant lacking the DNA binding domain continued to repress MT-I promoter. Overexpression of MTF-1, the key trails-acting factor involved in MT-I gene transcription, surmounted NFI-mediated repression of the basal and zinc-induced MT-I promoter activity. These data demonstrate that NFI is a repressor of MT-I expression, where its DNA binding activity is not essential to downregulate the MT-I promoter. Interaction of NFI with another protein(s), probably MTF-I, may be involved in this repression.