A technique for isolation of viable spermatozoa from the rat vas deferens is presented. A single vas deferens from a mature Sprague-Dawley rat contains 35.4 +/- 3.3 million spermatozoa, and a 0.5-cm segment from the distal end of the vas will spontaneously expel 8.96 +/- 1.39 million sperm relatively free of cellular debris when placed into a balanced salt solution. Treatment of rats (n = 5) with alpha-chlorohydrin (100 mg/kg, single dose) significantly reduced, relative to controls (n = 5, water 10 mL/kg), sperm motility by 72%, swimming velocity by 44%, and head displacement motion by 50%, but did not affect the number of sperm in the vas deferens nor sperm recovery. In summary, the isolation of rat sperm from the vas deferens can be achieved with minimum handling in a manner that is not confounded by sperm toxicity.