Background: Vitamin A and D analogues play an important role in epidermal homeostasis and are used in the treatment of various skin diseases. The failure of retinoid and vitamin D treatments is sometimes difficult to explain. Methods: We analyzed the effect of all-trans retinoic acid (all-trans RA), 13-cis retinoic acid (13-cis RA), ergocalciferol and cholecalciferol in keratinocyte cultures established from adult donors, on the cell proliferation by means of [3H]thymidine incorporation and apoptosis after fluorescein diacetate/trypan blue staining. Results: All tested agents exerted a dose-dependent inhibition of keratinocyte proliferation in the concentration range of 1.25–5 µM. Based on IC50 values, the antiproliferative efficiency was as follows: cholecalciferol > ergocalciferol = all-trans RA > 13-cis RA. The observed effect of cholecalciferol and ergocalciferol, but not retinoids, involved the induction of apoptotic cell death. Combining vitamins A and D did not further increase the proliferation block and even displayed an antagonistic effect. Conclusion: The susceptibility of keratinocytes to the antiproliferative action of vitamins A and D was markedly different in cell cultures derived from different donors, indicating a possible predictive value of the in vitro testing for the efficiency of the clinical response to these agents.