An attempt to improve the post-thawing quality of dog semen was made, using proline and glycine betaine in three different concentrations. These amino acids, used in combination and individually, showed cryoprotective properties when they were added to a ram semen extender, and this study was carried out to determine whether these compounds could also be effective for frozen canine sperm. In this study, semen from 13 dogs was pooled and frozen in a Tris-fructose-citric acid extender containing various concentrations of proline and glycine betaine, both alone or in combination. The following parameters were evaluated post thawing as indicators of semen quality: progressive motility, live:dead ratio and acrosomal integrity. Analysis of variance at 0, 0.5, 1, 2 and 4 h post-thaw indicated that proline, in all the evaluated concentrations, exerts a beneficial effect on the post-thaw canine spermatozoa motility and longevity, as well as on the acrosomal integrity. Glycine betaine alone or combined with proline, did not show any difference compared with the control samples for any of the concentrations used.