The rabbit is considered to be a valuable laboratory animal. We compared 2% acetamide and glycerol as cryoprotectants in egg-yolk diluent for ejaculated Japanese white rabbit spermatozoa to improve sperm cryopreservation methods. Fertility through artificial insemination, forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rates of forward progressive motility and plasma membrane integrity of the spermatozoa frozen with acetamide (27.1 +/- 8.3% and 24.5 +/- 6.5%) were significantly (P < 0.05) higher than those of the spermatozoa frozen with glycerol (16.3 +/- 10.9% and 14.3 +/- 7.6%). Though there was no significant difference in the kindling rates, the litter size of females inseminated with spermatozoa frozen with acetamide (6.0 +/- 1.1) were significantly (P < 0.05) higher than those of spermatozoa frozen with glycerol (3.0 +/- 0.4). The results indicate that 2% acetamide has a higher cryoprotective effect than 2% glycerol for sperm cryopreservation in the Japanese white rabbit.