Peroxisome proliferator-activated receptors (PPARs) are pleiotropic regulators of growth and differentiation of many cell types. We have performed a comprehensive analysis of the expression of PPARs, transcriptional cofactors, and marker genes during differentiation of normal human keratinocytes using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARdelta was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARalpha and PPARgamma expression was linked to differentiation, and accordingly, expression of PPARalpha and PPARgamma was in essence confined to suprabasal cells. Differentiation was not accompanied by significant changes in the expression of the coactivators CREB-binding protein, p300, steroid receptor coactivator 1, or the corepressors nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order PPARdelta >> PPARalpha > PPARgamma. All selective PPAR ligands marginally induced transglutaminase-1 expression with the PPARdelta-selective ligand L165041 being the most potent. The PPARalpha- and PPARgamma-selective ligands Wy14643 and BRL49653 had negligible effect on involucrin expression, whereas a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease in proliferation and a robust upregulation of the expression of involucrin and transglutaminase. Our results indicate that tetradecylthioacetic acid may affect keratinocyte gene expression and differentiation via PPAR-dependent and PPAR-independent pathways, and that the latter play an important role.