In the present study, effects of glycerol addition to semen at various temperatures and cooling rates of semen on the post-thaw semen characteristics on ram semen was investigated. Semen of 10 Kivircik rams of 3-5 years old were collected, pooled and frozen in straws. Six groups were established according to the extension of semen in a single step (30°C) or two steps (30°C and 5°C) and to the cooling technique to 5°C [slow (<0.2°C/min) or two (from 30°C to 16°C <2°C/min and then from 16°C to 5°C <0.2/min) stages]. Semen was extended with extender A without glycerol at 30°C and cooled to 5°C slowly (group 1) or in two-stages (group 2), at this temperature extender B was added. Again semen was extended with 2% glycerol bearing extender A at 30°C and cooled slowly (group 3) and in two-stages (group 4). The other groups were extended at 30°C with extender A and B (4% glycerol) and cooled slowly (group 5) or in two-stages (group 6). Samples were frozen in 0.25 ml straws after the equilibration. Spermatological examinations (motility, acrosomal and total morphological defect rates) were done after extension (level 1), after cooling to 5°C (level 2), after equilibration (level 3) and after thawing (level 4). In the present study, it was concluded that (1) cooling from 30°C to 5°C in the presence of glycerol at 1:1/2 dilution rate, deteriorated the post-thaw motility, (2) this deterioration was compensated by two-staged cooling technique.