To screen genes involved in P15NK4b regulation during cell cycle, differential display method was applied to compare mRNAs from G1 synchronized cells of MLIK6, which overexpressed P15INK4b gene, and itscontrol MLC2. By using thisapproach, 15 cDNA fragments that were preferentially expressed in MLIK6 cells, but not in MLC2 cells, were screened out. A novel gene named P15RS was identified with further analysis. Combining the sequence from DD-PCR, homology analysis against EST database and RACE, a 4404 bp complete cDNA sequence of P15RS was generated. Sequence analysis revealed that P15RS cDNA encoded a 312-aminoacid peptide containing a RAR domain that is involved in regulation of nuclear pre-mRNA, which suggests that P15RS may be a nuclear regulation protein. Genomic sequence analysis demonstrated that human P15RS gene was localized on chromosome 18q12 with seven exons and six introns. Expressing antisense P15RS in MLIK6 cells can up-regulate the expression of cyclinD1 and cyclinE. These data indicate that P15RS may act as a negative regulator in G1 phase.