The permeability of murine cauda epididymidal spermatozoa was determined from the swelling caused by penetrating agents at isotonicity, which lies between 422 and 530 mmol/kg. Spermatozoa were permeable to a range of solutes with size !200 Da. Relative entry rates of cryoprotective agents (CPAs) were ethylene glycolzDMSOOpropane-1,2-diolOglycerolOpropane-1,3-diol. More polar compounds including major epididymal secretions were impermeant. None of the compounds entered spermatozoa through quininesensitive channels; rather, quinine increased the size of solute-swollen spermatozoa, suggesting that regulatory volume decrease and osmolyte loss occurred under these conditions. Volume responses to lowered osmolality revealed a greater volume-regulating ability of spermatozoa from the B6D2F1 strain than the C57BL6 strain. As the former strain displays better post-thaw fertility, their spermatozoa may have greater osmolyte loads enabling them to cope better with osmotic stress. Inadequate volume regulation, due to CPA-induced osmolyte loss, may affect post-thaw fertility. Knowing the permeability towards cryoprotectants will help to make a better choice of CPAs that are less damaging to sperm during cryopreservation. Reproduction (2008) 135 625-633