Analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)2D3) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1alpha,25(OH)2D3 and its analog EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensitivities to 1alpha,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D3 receptor (VDR) mRNA and protein, and no significant variation was observed in 1alpha,25(OH)2D3-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1alpha,25(OH)2D3-sensitive reporter gene. The antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes on two cDNA microarrays, yielding 38 up-regulated targets, including adhesion molecules, growth factors, kinases, and transcription factors. Genes encoding factors implicated in cell cycle regulation were induced, including the growth arrest and DNA damage gene, gadd45alpha, and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction of GADD45alpha protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. While 1alpha,25(OH)2D3 and EB1089 modestly induced transcripts encoding the cyclin-dependent kinase inhibitor p21(waf1/cip1), no changes in protein levels were observed, indicating that p21(waf1/cip1) induction does not contribute to the antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there was extensive loss of target gene regulation (10 of 10 genes tested), indicating that resistance arises from widespread loss of 1alpha,25(OH)2D3-dependent gene regulation in the presence of normal levels of functional VDRs.