ABSTRACT: Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75–900 mOsmol kg−1), as shown by the Boyle van't Hoff plot (r2 = .99). Rhesus spermatozoa have a mean cell volume of 36.8 ± 0.5 μm3 at 22°C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integrity to deviations from isotonicity and, in addition, that rhesus sperm motility and membrane integrity were more sensitive to hypertonic than hypotonic conditions. Mitochondrial membrane potential did not explain the lack of sperm motility observed under anisosmolal conditions in our study. Although most spermatozoa were able to recover initial volume after osmotic stress, they were not able to recover initial motility.