Cryopreservation of avian semen is a useful tool to preserve genetic resource for aim of preventing extinction induced by infectious disease like avian influenza. Unlike those of mammals, data from chicken cryopreserved semen has not been showed feasible results. So, various cryoprotectants and diluents have been examined in many methods. In this report, as a major ingredient of avian seminal plasm, glutamine was substituted by alanyl glutamine to enhance physiological stability of chicken semen during freezing. We studied effect of glycerol and Dimethylacetamide(DMA) on motility and progressive motility of spermatozoa using glutamine diluent(EK-G) or alanyl glutamine diluent(EK-A) condition. The semen of Ogye was collected twice a week by the dorso-abdominimal massage method and diluted with same volume of EK-G or EK-A at 25℃ and stored for 10 min at 4℃ in cold chamber. Glycerol or DMA was added to diluted semen to reached 7% of final concentration at 4℃. After 3min of equilibration, the diluted semen was packed into 0.25ml straws and subjected to cryopreservation used freezing equipment. The packed straw were placed on height 5 cm above surface of liquid nitrogen(LN2) and held for 10min. After preserved for 2 weeks, the straw was thawed onto the 4℃ cooling bath. The images of motility and progressive motility spermatozoa were recorded by digital image recorder and analyzed by manual. The results showed 68.5% motility and 34.1% progressive motility in DMA/EKA diluent, 31.45% and 17.6% in glycerol/EKA, 45.4% and 8.6% in DMA/EKG, and 9.7% and 6.4% in glycerol/EKG. With these results, the alanyl glutamine and DMA could be used as a main composition of diluent and cryoprotectant for cryopreservation of chicken semen.