We and others have previously reported that transforming growth factor-α (TGFα) expression is hormonally responsive and its expression is coregulated with that of its receptor [the epidermal growth factor (EGF) receptor]. The 5′-flanking region of the TGFα gene was characterized to determine whether it could confer hormone responsiveness to a reporter gene (luciferase) in human mammary carcinoma cells (MDA468). This segment of the gene is GC rich and contains an element strikingly similar to the core element of the EGF receptor gene that has been shown to mediate both basal and hormonestimulated expression of the EGF receptor. We now report that a 313-basepair (bp) proximal element of the TGFα 5′-flanking region (−373 to −59 relative to the TGFα translation start codon) is capable of conferring responses to phorbol ester and EGF. This gene segment does not contain the EGF receptor gene homolog or potential AP-2-binding sites, suggesting that these elements are not necessary for basal and EGF- or phorbol ester-responsive TGFα gene expression. This 313-bp proximal element also confers proper transcriptional initiation to the chimeric TGFα-luciferase reporter construct, indicating it is the TGFα promoter. A 1.1-kilobase segment of the TGFα 5′-flanking region also confers retinoic acid, thyroid hormone, and glucocorticoid responsiveness despite the absence of recognizable steroid hormone receptor-binding sites. These hormones stimulate reporter expression 1.5- to 2-fold in a dose-dependent manner. Extension of the 5′-flanking region to −3500 results in marked suppression of reporter gene expression. These results indicate that the TGFα gene 5′-flanking sequence contains the elements responsible for hormonal responsiveness of this gene and that these elements are distinct from those that regulate the expression of the EGF receptor gene. © 1991 by The Endocrine Society.