This study was conducted to determine the osmotic properties of bull spermatozoa, including the effects of osmotic stress and cryoprotectant agent (CPA) addition and removal, on sperm motility. Semen from beef bulls was collected by electroejaculation and extended 1:3 in TL-Hepes containing 100 μg/ml pyruvate and 6 mg/ml BSA. In solutions of 150–1200 mOsmolal (mOsm), bull spermatozoa behaved as linear osmometers (r² = 0.97) with an osmotically inactive cell volume of 61%. The isosmotic cell volume was 23.5 μm³. Motility was determined after exposure to anisosmotic solutions ranging from 35 to 2400 mOsm and after return to isosmotic conditions. Retention of at least 90% of isosmotic motility could be maintained only between 270–360 mOsm. Bull spermatozoa were calculated to retain 90% of their isosmotic motility at 92–103% of their isosmotic cell volume. Motility following a one-step addition and removal of 1 M glycerol, dimethyl sulfoxide, and ethylene glycol was reduced by 31%, 90%, and 6%, respectively, compared with CPA addition only. These data indicate that, during bull spermatozoa cryopreservation, osmotically driven cell volume excursions must be limited by exposure to a very narrow range that may be facilitated by the use of ethylene glycol as a CPA.