Background: Cultured epidermal sheet (CES) grafts accelerate wound healing as a result of so-called biological dressing effect, which is thought to be mediated by various growth factors. However, the profile of growth factor expression in CESs is unclear. Objective: To investigate whether CESs produce growth factors along with stratification we investigated the production of growth factors and their regulation in CESs. Methods: CESs conditioned medium was harvested and the concentration of TGF-α, TGF-β1, TGF-β2, and VEGF was measured using ELISA. The mRNA of EGF family, TGF-β family and VEGF was detected by Northern blot or RNase protection assay. Results: The concentration of TGF-α was 100 pg/ml in the monolayer culture, but dramatically increased to 600 pg/ml 2 days after stratification. It decreased to baseline, and then gradually increased to 300 pg/ml in the presence of EGF and remained at that level until day 20. TGF-β1 increased from 50 to 400 pg/ml after stratification, and remained at that level day 20. TGF-β2 was undetectable in the monolayer culture, but dramatically increased to 200 pg/ml 2 days after stratification. Unlike TGF-β1, TGF-β2 gradually increased over time after stratification. VEGF increased with stratification from 500 to 1500 pg/ml. The addition of EGF upregulated EGF family, TGF-β, and VEGF production in CESs, as confirmed by ELISA, Northern blot, and RNase protection assay. Conclusion: These results indicate that so-called biological dressing effect of CESs is mediated by production of the EGF family, TGF-β, and VEGF. Our results also demonstrate the ability of EGF to enhance growth factor production in CESs.