Four different concentrations of glycerol in a Tris-fructose-citric acid extender for frozen dog semen and the effects of adding glycerol at 37 degrees C or 4 degrees C to the extender were studied by monitoring the post-thaw sperm longevity and acrosomal integrity during incubation at 39 degrees C. In the first part of this study, ejaculates from 13 dogs were pooled and divided into 4 aliquots, which were centrifuged and the sperm pellets rediluted with a Tris-fructose-citric acid extender containing 2, 4, 6 and 8% (v/v) glycerol, respectively. Progressive motility by subjective estimation, live:dead spermatozoa ratio using eosin-nigrosin staining, and acrosomal integrity using phase contrast microscopy were evaluated before processing and at 0, 0.5, 1, 2 and 4 hours post-thawing incubating the semen samples in the dark at 39 degrees C. The experiment was performed using seven replicates and it was found that sperm motility and acrosomal integrity were superior following the use of 8% glycerol in the extender. In Experiment 2, 13 ejaculates from the same dogs used in the first experiment were pooled and divided into 3 aliquots, and an 8% glycerol diluent was added at 37 degrees C and 4 degrees C after 1 h of cooling or at 4 degrees C after 2 h of cooling, respectively. After freezing and thawing the same parameters as studied in the first experiment were assessed. The experiment was performed in 7 replicates, and no difference was found between treatments.