We previously reported that hormones important for the normal growth and function of FRTL-5 rat thyroid cells, thyrotropin (TSH) or its cAMP signal plus insulin or insulin-like growth factor-1 (IGF-1), could transcriptionally suppress constitutive and -interferon (IFN)-increased synthesis of the 90K protein (also known as Mac-2BP). Here we cloned the 5'-flanking region of the rat 90K gene and identified a minimal promoter containing an Interferon Response Element (IRE) and a consensus E-box or USF binding site, which are highly conserved both in the human and murine genes. We show that suppression of constitutive and -IFN-increased 90K gene expression by TSH/cAMP plus insulin/IGF-1 depends on the ability of the hormones to decrease the binding of USF to the E-box, located upstream of the IRE. This site is required for the constitutive expression of the 90K gene. Transfection with USF1 and USF2 cDNAs increases constitutive promoter activity, attenuates the ability of TSH/cAMP plus insulin/IGF-1 to decrease constitutive or -IFN-increased 90K gene expression, but does not abrogate the ability of -IFN itself to increase 90K gene expression.