In an attempt to optimize a method of cryopreserving spermazoa from mice bearing mutations, we investigated the effect on motility of temperature for the collection of mouse sperm and the rate of thawing after freezing. Comparison among samples of sperm collected from both the caudae epididymides and vas deferens placed directly in a cryoprotectant of 3% skim milk and 18% raffinose equilibrated at 37, 23, and 3 degrees C showed no difference in the number of viable sperm harvested. Concentration and motility was highest after collection at 37 degrees C (22.3 x 10(6) sperm/ml with 80% motility) combined with rapid thawing at 37 degrees C (2.9 x 10(6) sperm/ml with 84% motility in the swim-up fraction). The fertilization capacity of sperm collected and thawed at 37 degrees C was analyzed in vitro and no difference was observed between the cryopreserved sperm and the control (91 and 89%, respectively). Transfer of in vitro fertilized embryos to pseudopregnant recipients resulted in 37% implantation at Day 10 of pregnancy and 38% live births at term.