In order to improve the effectiveness in the cryopreservation of horse sperm, the effect of association L-glutamine with Ethylenglicole and Glycerol in the freezing media spermatozoa was evaluated. 4 Colombian native stallions were used to complete a total of 21 samples which were frozen in two different media: INRA 97 and cryoprotectant. The following study was done: L-glutamine 80mM + Etilenglicol 2.5% (protocol 1), L-glutamine 80 mM + Glycerol 2.5% (protocol 2), Etilenglicol 2.5% (protocol 3) and glycerol 2.5% (protocol 4). The freezing methodology was: 60 minutes to descend the temperature from 38°C to 5°C (0.55°C/min) during the transport. The samples were centrifuged at 600G/10min., and the semen was diluted with the four protocols in straws of 0.5 ml. Then, 60 minutes of equilibrium in refrigeration; 20 minutes in liquid nitrogen vapors and then immersed. In the progressive motility evaluation there was not any significant difference between protocols at 0 time (p ≤ 0.6383), at 30 minutes (p ≤ 0.511), and at 60 minutes (p ≤ 0.1659). The motility averages for the 4 protocols at 0 time were (1) 29,6 ± 15,1; (2) 28,1 ± 13,5; (3) 28,4 ± 12,3 and (4) 38 ± 11,1; at the 30 minutes: (1) 25,1 ± 13,6; (2) 22,3 ± 13,0; (3) 24,9 ± 12,4 and (4) 25,5 ± 11,6, and at 60 minutes (1) 17,1 ± 12; (2) 15,4 ± 11,7; (3) 19,9 ± 11,5 and (4) 17,6 ± 14. The spermatic survival was evaluated with eosine-nigrosine coloration, after thawing and there was not any significant difference among the protocols (p≤ 0.6336), the average measures were (1) 37; (2) 28,8; (3) 28,7 and (4) 31,7. As a conclusion, although significant difference was not demonstrated among the protocols; the tendency to the highest average was presented by the protocol 4 (glycerol 2.5%).