Analogues of vitamin D3 have recently been introduced for the topical treatment of psoriasis. Their therapeutic effects are thought to be mediated by interaction with the vitamin D3 receptor (VDR) in epidermal keratinocytes (KCs). The purpose of the present study was to investigate the trans-acting activity of the endogenous VDR in human KCs transfected with a vitamin D response element (VDRE) in response to 1,25-dihydroxývitamin D3 [1,25(OH)2D3] and the synthetic vitamin D3 analogues GS 1500, EB 1213, MC 903 (calcipotriol) and KH 1060. Cultured KCs obtained from normal human adults were transfected with a VDRE consisting of a direct repeat (DR) of 2 hexanucleotides separated by 3 nucleotides (AGGTCAaggAGGTCA) cloned as a triple copy into the chloramphenicol acetyltransferase (CAT) reporter plasmid pBLCAT2. This DR3 response element is preferentially activated by heterodimers of the VDR and the retinoid X receptor. Twenty-four hours after transfec-tion, 1,25(OH)2D3, vitamin D3 analogues or 9-cis-retinoic acid (9-cis-RA) were added, and, after an additional 24 h, cells were harvested and assayed for CAT activity. 1,25(OH)2D3 dose-dependently induced CAT activity in VDRE-transfected KCs and co-transfection with exogenous human VDR enhanced the response to 1,25(OH)2D3. Induction of CAT activity by 1,2 5 (OH) 2D3 was enhanced in the presence of the endogenous ligand for retinoid X receptor, 9-cis-RA. The synthetic vitamin D3 analogues dose-dependently stimulated CAT activity. Compared to 1,25(OH)2D3, KCs were less sensitive to stimulation with MC 903, equally sensitive to EB 1213 and more sensitive to GS 1500 and KH 1060. In conclusion, the endogenous VDR in KCs is responsive to 1,25(OH)2D3 and its synthetic analogues in stimulating gene transcription. To the extent that the biological actions of vitamin D3 are dependent on its ability to induce gene transcription through the endogenous VDR, this transfection model may be used in the screening of novel vitamin D3 analogues for biological activity.