Abstract: Effects of five TEST extenders varying in osmolality on rhesus monkey sperm cryopreservation were studied,using TTE as the control. These five extenders were designated as TEST,mTEST1,mTEST2,mTEST3 and mTEST4,and the corresponding osmolalities were 688,389,329,166 and 43 mOsm/kg,respectively. Semen were diluted in one step into freezing media containing glycerol,and the final concentration of glycerol was 5%(v/v). Sperm survival was determined by assessment of sperm motility and membrane integrity prior to and after cryopreservation. Membrane integrity was evaluated using Hoechst 33342/propidium iodide dual staining procedure and flow cytometry. The results showed that,before freezing,motility and membrane integrity of spermatozoa diluted in TEST and mTEST4 were drastically reduced (P<0.001) compared with fresh sperm. In remaining groups,sperm function remained unaffected after equilibration except that the membrane integrity of spermatozoa diluted in mTEST2 was significantly lower than that of fresh semen (P<0.05). The sperm cryopreservation efficiency for these extenders,evaluated by post-thaw sperm motility and membrane integrity,is: TTE,mTEST3 and mTEST1> mTEST2> TEST and mTEST4 (P<0.05). The results suggest that isosmotic,moderately hyperosmotic or hypoosmotic extenders are favorable for the cryopreservation of rhesus monkey spermatozoa and the hyperosmotic stress of TEST accounts for the lower survival of rhesus monkey spermatozoa frozen in this medium.