Three levels of glycerol were added to chicken semen to determine its effect on trypsin-like enzyme activity (TLE) and fertility of spermatozoa. Fertility was significantly depressed by 0.5, 2.0 and 6.0% glycerol, but sperm motility was not affected. Trypsin-like activity was reduced in glycerol treated spermatozoa, but the reduction in sperm TLE activity did not correspond to the severe reduction in the fertilizing capacity. Electrophoretic fractionation of TLE in polyacrylamide gels revealed two fractions of TLE in chicken spermatozoa. The mechanism for the fertility depressing action of glycerol on chicken spermatozoa apparently does not involve the TLE of the sperm acrosome.