The permeability of human spermatozoa to glycerol and its activation energy were determined using electron paramagnetic resonance (EPR) techniques. EPR was used to monitor the aqueous cell volume change vs. time during the glycerol permeation process using the aqueous spin label 15N- tempone and the membrane impermeable broadening agent potassium trioxalatochromiate (chromium oxalate). The permeation process was completed in tens of seconds, requiring the use of a stopped-flow methodology. The glycerol permeability coefficient ( P g ) was determined by fitting a simple theoretical model to the experimental data. The permeabilities of human spermatozoa in 1 molar and 2 molar glycerol at 20°C are (10.3 ± 0.3) · 10 −4 cm/min ( mean ± S.D. ) and (6.0 ± 1.4) · 10 −4 cm/min , respectively. The permeabilities of human spermatozoa in 2 molar glycerol at 30, 20, 10, and 0°C are (8.3 ± 1.3) · 10 −4 cm/min , (6.0 ± 1.4) · 10 −4 cm/min , (2.1 ± 0.4) · 10 −4 cm/min , and (1.1 ± 0.3) · 10 −4 cm/min , respectively. The activation energy ( E a ) for glycerol permeation between 30°C and 0°C was found to be 11.6 kcal/mol.