The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH)(2)D(3) in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH)(2)D(3) treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12h after treatment with 1,25-(OH)(2)D(3) (10(-8)M), preceding (6h) or coinciding with (12h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12h incubation period with 1,25-(OH)(2)D(3) were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH)(2)D(3) was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH)(2)D(3) resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest.