All-trans retinoic acid (RA) compromises epidermal differentiation and causes keratinocyte hyperproliferation through mechanisms not completely understood, but may involve the regulatory matrix molecule hyaluronan. In this work, the influences of all-trans RA on epidermal morphology and hyaluronan metabolism were examined in organotypic and monolayer cultures of rat epidermal keratinocytes (REKs). All-trans RA treatment of organotypic REK cultures (10 days) increased the synthesis of hyaluronan, the expression of hyaluronan synthases Has2 and Has3, and the CD44 receptor, with hyperplasia of the epidermis. The hyperplasia and hyaluronan production induced by all-trans RA were blocked with (1) AG1478, an inhibitor of the EGFR; (2) UO126, an inhibitor of the MAPK/ERK kinase, and (3) GM6001, an inhibitor of the matrix metalloproteinases. These effects were consistent with the findings that all-trans RA upregulated heparin-binding epidermal growth factor-like growth factor mRNA expression and increased the phosphorylation of EGFR and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, the activation of EGFR and ERK1/2 was seen already 30 minutes after all-trans RA treatment, suggesting that the activation of this signaling pathway is a primary response to all-trans RA. These results indicate that the effects of all-trans RA on keratinocyte proliferation and hyaluronan synthesis are partly mediated through EGFR signaling.