Retinoic acid (RA) has been shown to be effective in eradicating premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck (SCCHN) in clinical trials. The basis for this effect is unclear. We have previously demonstrated that messenger RNA from tumor growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGFR), is unregulated in tumors and histologically normal mucosal samples from patients with SCCHN compared with control normal mucosa from patients without cancer, implicating this ligand-receptor pair in an autocrine growth pathway early in the molecular pathogenesis of this disease. In this report, we examined the hypothesis that the action of RA on the mucosa of the upper aerodigestive tract is mediated via downregulation of steady-state TGF-alpha and/or EGFR mRNA levels. Following exposure to all-trans-RA, a series of SCCHN cell lines demonstrated a 35.4% reduction in TGF-alpha mRNA expression (P = 0.022) and 58.5% reduction in EGFR mRNA (P = 0.0027). Nuclear run-on analysis indicated that the RA-mediated reduction of TGF-alpha and EGFR steady-state mRNA levels was a result of decreased gene transcription. These results suggest that the clinical effects of RA in SCCHN patients may be due to a downmodulation of TGF-alpha and EGFR mRNA production.