The objective of the study was to evaluate the timing of adding glycerol during the cooling process on sperm quality of canine semen. Fifteen ejaculates from 4 mature dogs were used. Each sample was divided in aliquots and three methods of adding glycerol were used (T1: at the beginning; T2; at the beginning and at the end; T3: at the end of the cooling process). Semen samples were packed in 0.5 ml straws and frozen in liquid nitrogen. The post-thawing progressive motility and the functional integrity of membrane were evaluated. None statistical difference was observed due to the three treatments, indicating that the process of cryopreservation can be simplified by adding all the glycerol at the beginning of the cooling curve.