Because a protective barrier is essential for life, the development of the epidermis and stratum corneum must be completed prior to birth. The epidermal permeability barrier is comprised of corneocytes embedded in a lipid enriched matrix. Recent studies from our laboratory, using an explant model of fetal rat skin development that closely parallels in utero development, have shown that hormones and other activators of members of the nuclear receptor family regulate permeability barrier ontogenesis by stimulating lipid metabolism and the formation of the extracellular lipid lamellae. Using this model we sought to determine whether these hormones and nuclear activators also regulate keratinocyte differentiation during fetal development. Profilaggrin/filaggrin and loricrin expression, assessed by in situ hybridization and by immunohistochemistry, were progressively increased during epidermal ontogenesis. Whereas profilaggrin/filaggrin and loricrin were not expressed at day 17 of gestation, by day 19 both were present in the upper layers of the epidermis and both became still more abundant by day 21. These developmental changes also occurred in fetal skin explants cultured in vitro for 4 d, although the expression levels did not appear as robust as in utero. Whereas neither profilaggrin/filaggrin nor loricrin were expressed in control explants cultured for 2 d, they were seen in explants treated with either thyroid hormone, glucocorticoids, or estrogens. In contrast, dihydrotestosterone treatment delayed the expression of profilaggrin/filaggrin and loricrin. Moreover, both clofibrate, a peroxisome proliferator-activated receptor-alpha ligand, and juvenile hormone III, a farnesoid X-activated receptor activator, markedly accelerated fetal epidermal differentiation, stimulating both profilaggrin/filaggrin and loricrin expression. Our results demonstrate that several hormones and activators of nuclear hormone receptors regulate epidermal differentiation during fetal development, affecting key constituents of both keratohyalin granules and the cornified envelope. Thus, a variety of ligands/activators of nuclear receptors accelerate not only permeability barrier ontogenesis, but also the expression of structural proteins essential for stratum corneum formation.