To gain more insight in the role of the vitamin D system in epidermal differentiation, we studied the expression of the vitamin D receptor and its heterodimeric partner retinoid X receptor alpha in cultured normal human keratinocytes during squamous differentiation, as triggered by different approaches. Northern and western blot analysis allowed us to investigate mRNA and protein levels of these nuclear receptors and of markers for growth control (c-myc, cyclin D1, p21WAF1) and differentiation (keratinocyte transglutaminase, small proline rich proteins). Growing cells to postconfluence was a potent stimulus for growth arrest and differentiation with concomitant suppression of vitamin D receptor and induction of retinoid X receptor alpha, at both the mRNA and the protein level. These changes could be prevented by concomitant treatment with epidermal growth factor or keratinocyte growth factor. Subjecting the cells to a calcium switch leading to stratification and differentiation lowered vitamin D receptor protein levels without affecting vitamin D receptor mRNA and induced both retinoid X receptor alpha mRNA and protein. Interferon-gamma and the phorbolester 12-O-tetradecanoyl phorbol 13-acetate, two well-known inducers of keratinocyte differentiation, both inhibited vitamin D receptor expression but only interferon-gamma induced retinoid X receptor alpha. The decreased vitamin D receptor expression was accompanied by reduced vitamin D responsiveness (as assessed by 24-hydroxylase mRNA induction) in postconfluent, high calcium, and 12-O-tetradecanoyl phorbol 13-acetate treated keratinocytes but not with interferon-gamma treatment. Taken together, our results associate vitamin D receptor expression with undifferentiated, proliferating keratinocytes, whereas retinoid X receptor alpha expression appears to be related to the differentiated phenotype. Therefore, proliferating and differentiating keratinocytes may be differentially targeted by active vitamin D metabolites.