Using human cyclin D1 as the "bait" in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1. This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle. However, the level of this protein increases severalfold during the differentiation of HL60 cells. The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle. DIP1 contains a helix-loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins. The dip1 gene is located on human chromosome 16p11.2-12, a locus that is amplified in several types of human cancer. These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined.