A DNA microarray analysis was carried out to examine changes in gene expression associated with 1α,25-dihydroxyvitamin D₃ (DVD)-mediated differentiation of HL-60 cells. Most of the genes for interleukins and interleukin receptors were up-regulated or remained unchanged in their expression upon differentiation. Among protein kinases C and A, the gene expression of protein kinase C beta 1, and to a lesser extent, protein kinase C alpha, was up-regulated by DVD. The gene expression of 8 amino acyl-tRNA synthetases was down-regulated >2 fold, and that of 9 was either down-regulated slightly or unchanged. Changes for some transcription factors and selected proteins are also discussed. These findings may help to reveal the molecular mechanism by which DVD acts in cellular events such as differentiation, tumor suppression, neuronal development, and immune modulation.