To compare the impact of multi- and single-step addition of cryoprotectants on human spermatozoa.Human semen was subjected to cryoprotective agents (CPAs) in both one-step and multistep protocols before removal of the CPAs by multi-step and single-step methods.Infertility clinic.Healthy normozoospermic volunteers.Single- and multistep addition and removal of a range of CPAs.Analysis of tail morphology and total and progressive motility, as well as kinematic parameters, in medium and sperm penetration into artificial mucus.While no CPA drastically affected sperm vitality, some were more deleterious than others. Propane-1,3-diol was the least damaging to sperm function, whereas glycerol and ethyl(hydroxymethyl)propane were particularly disruptive. For all CPAs, multistep addition and removal of CPAs proved less damaging to sperm function than the single-step protocol.Optimal CPAs may be those that minimize osmotic stresses to spermatozoa during prefreeze and post-thaw procedures.