A protocol was developed for the separation, concentration enumeration, and cryopreservation of Neospora caninum tissue cysts from mouse brains. Brains from chronically infected mice were homogenized and tissue cysts counted in 10-microliters aliquots. Tissue cysts were separated from brain homogenates by centrifugation at 4,400 g on 35% (v/v) Percoll/phosphate-buffered saline (PBS) continuous-density gradients. After removal of the brain layer, the separated tissue cysts were concentrated by diluting the remaining solution with PBS and centrifuging at 500 g. The pellet was resuspended in PBS and tissue cysts were enumerated. Fifty percent of tissue cysts were recovered from brains centrifuged once and 64% from brains centrifuged twice. Tissue cysts were preserved with 7.5% dimethyl sulfoxide in horse serum at -60 C. After thawing, bradyzoites were digested in an acid/pepsin solution and placed onto Vero cell cultures. Neospora caninum tachyzoites were recovered from cell cultures, indicating that bradyzoites retained viability after concentration and cryopreservation. Separated tissue cysts ranged in diameter from 107 microns to 15 microns (average = 31 microns), and the average bradyzoite dimensions were 2 x 7.5 microns. These methods make it possible to store viable N. caninum tissue cysts for oral-infectivity trials and other studies.