Vitamin D(3) promotes myeloid leukemic cell lines to differentiate terminally into monocytes/macrophages. It has been reported that overexpression of the cdk inhibitor p27(Kip1) results in the differentiation of the myelomonocytic U937 cell line and that this gene is the target of vitamin D(3). To identify the sequences required for the positive regulation of p27(Kip1) transcription by vitamin D(3), a 3.6-kilobase 5'-flanking region of the human p27(Kip1) gene was examined by transiently transfecting luciferase reporter constructs into U937 cells. The transcriptional activity of this construct was activated by vitamin D(3). Deletion and mutational analysis revealed that both a GGGCGG sequence (-545/-539) and a CCAAT sequence (-525/-520) were necessary to induce p27(Kip1) gene expression. Importantly, the region containing both of these elements conferred positive responsiveness to vitamin D(3) to a heterologous promoter. Gel shift assays showed that Sp1 binds to the GGGCGG sequence and that NF-Y binds to the CCAAT sequence. Consistent with the roles of these transcription factors, treatment with vitamin D(3) stimulated the DNA binding activities of these factors to each element and induced the change of one NF-Y subunit. We conclude that vitamin D(3) stimulates transcription of the p27(Kip1) gene by a novel mechanism involving Sp1 and NF-Y, but not the vitamin D receptor, during the early stages of U937 cell differentiation.