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The natural cytotoxicity receptor NKp44 is an important regulator of NK cell function. We have previously reported that NKp44 has binding affinity to heparin and the transferrin containing multimeric sialyl Lewis X produced by human hepatoma HepG2 cells (HepTF) is more highly expressed than the other natural cytotoxicity receptors. To further understand the differences between natural cytotoxicity receptors, we sought to understand the gene transcriptional regulation of NKp44 in KHYG-1 human NK leukemia cells. 5′-rapid amplification of cDNA ends revealed the location of the NKp44 transcription initiation sites allowing identification of the 5′ untranscribed region. The result of the dual luciferase assay suggested that regulation of NKp44 gene expression is affected by the -1963 to -1599 nt region and the -352 to -231 nt region relative to the transcription initiation sites. The -410 to +1 nt region contained potential binding sites for the transcription factors AP-1, Oct-1, HNF-4, and Pax-4. These results suggest that defined regions in the 5′-flanking region of NKp44 gene is important for NKp44 transcription and regulation.