Human bronchial epithelial (HBE) cells undergo squamous differentiation in response to a variety of conditions in tissue culture, and retinoid treatment has been shown to reverse this process. Retinoids mediate their effects through the retinoic acid and retinoid X nuclear receptors (RAR and RXR, respectively), which form RAR-RXR heterodimers, RXR homodimers, and heterodimers of RXR and certain orphan receptors. These receptor dimers bind to distinct response elements, activating separate pathways. In this study, we investigated the roles of RAR and RXR signaling pathways in the inhibition of HBE squamous differentiation. After induction of squamous differentiation by confluent growth, HBE cells were treated with retinoids that selectively activate RARs (E-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthaienyl)-1- propenyl] benzoic acid), RXRs (4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid), or both RARs and RXRs (9-c/s retinoic acid). These retinoids inhibited the mRNA expression of the squamous differentiation markers transglutaminase type I, involucrin, keratin 5, and keratin 13, suggesting that inhibition of HBE squamous differentiation could be mediated by activation of either RAR or RXR signaling pathways. We examined the role of AP-1 as a potential effector of these retinoid pathways. AP-1 transcriptional activity was reduced markedly by these retinoids, and a concomitant, but proportionally smaller, reduction in AP-1 DNA binding was observed. Furthermore, treatment of squamous HBE cells with the retinoid SR11238, which inhibited AP-1 without activating retinoid receptor transcriptional properties, reduced the expression of transglutaminase type I and involucrin. These findings support the hypothesis that, in HBE cells, RAR and RXR signaling pathways inhibit AP-1 transcriptional activity, and this contributes to retinoid-induced reversal of HBE squamous differentiation.