Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from -1645 to -1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region a fragment from -1645 to -1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from -1605 to -1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] -1635 to -1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt -1599 to -1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.