Objective To investigate the possibility that the construction and expression of a eukaryotic expression vector system of rat NNNAT gene. Methods The fulllength cDNA fragment of rat AANAT gene was amplified by RTPCR method. After retrieving the PCR products, ligating it with pTARGETTM vector, transformating ligation reaction to JM109 huge efficiency competent cells and identifying the recombinant plasmid, the recombinant eukaryotic expression vector pTARGETTM-AANAT was transfected into rat L6 myoblasts with lipofectamine. Accordingly, engineered cells selected by antibiotic G418 were detected by the methods of RT-PCR and Westem blotting. Results It was revealed that, amplified AA-NAT cDNA confirmed by agarose gel electrophoresis could ligate with pTARGETTM vector and subcloned into JM109 cells. L6 cells transfected with pTARGETTM-AA-NAT survived well after G418 selection and expressed AA-NAT protein. Conclusion Our results suggest that we have prepared rat AA-NAT stable eukaryotic expression system successfully although it was just a primary result. This system can be used for th