We studied the effect of all-trans retinoic acid (all-trans-RA), 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) on proliferation and differentiation of human keratinocytes cultured in a submerged culture system for up to 5 weeks and evaluated changes in cell morphology and in the expression of proliferation- and terminal differentiation-related genes on both the mRNA and the protein levels. Under control culture conditions, the expression of small proline-rich proteins (SPRR1 and SPRR2), involucrin, Ki67 and c-jun reached a maximum after 2 weeks in culture (1 week postconfluence) and then decreased as the tissue architecture of the cultures deteriorated. Upon simultaneous treatment with both retinoids and 1,25(OH)2D3 a culture was generated that remained stable for 4 weeks with at least eight living cell layers. Furthermore, this culture showed a pattern of SPRR2 and involucrin expression which closely resembled that of native epidermis, a maintained Ki67 expression and a strongly induced c-jun expression. Treatment with 1,25(OH)2D3 alone inhibited cell proliferation and stimulated cell differentiation resulting in acceleration of the differentiated phenotype and was accompanied by inhibition of c-jun and Ki67 expression and also, surprisingly, by inhibition of SPRR1, SPRR2 and involucrin expression. In contrast, treatment with all-trans-RA and/or 9-cis-RA induced a more proliferative phenotype with a prolonged lifespan as compared to control cultures. SPRR1 was weakly repressed, SPRR2 was strongly repressed, a delayed induction of involucrin occurred, and c-jun and Ki67 expression were maintained. These results show that modulation of the composition of the medium by the addition of various vitamins results in changes in the balance between keratinocyte proliferation and differentiation which correspond to changes in the expression of proliferation and differentiation markers and prolongation of the culture lifespan.