Nuclear retinoic acid receptor β (RARβ) expression is suppressed in many head and neck squamous cell carcinomas (HNSCCs), and an inverse relationship was found between squamous differentiation and RARβ expression in such cells. To investigate the role of RARβ in HNSCC growth and differentiation, we transfected a retroviral RARβ₂ expression vector (LNSβ) into HNSCC SqCC/Y1 cells, which do not express endogenous RARβ but do express RARα, RARγ, and retinoid X receptors. Transfected clones expressing RARβ₂ mRNA and protein exhibited enhanced sensitivity to the suppressive effects of all-trans-retinoic acid (ATRA) on squamous differentiation compared with cells transfected with the LNSX vector only; transglutaminase type I level was suppressed after a 3-day treatment with 10⁻¹⁰ m ATRA in four of five LNSβ clones, whereas it was not suppressed in LNSX cells even by 10⁻⁶ m ATRA. Similarly, cytokeratin 1 mRNA level was more suppressed in ATRA-treated LNSβ clones than it was in LNSX cells. This effect was independent of transrepression of activator protein-1. None of the LNSβ-transfected clones showed an increased growth inhibition by ATRA, 9-cis-retinoic acid, or the synthetic retinoid 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid. These findings suggest that, in SqCC/Y1 cells, RARβ mediates suppression of squamous differentiation by ATRA without enhancing its growth-inhibitory effects.