Although numerous techniques are available for the isolation of spores from myxosporidan and microsporidan parasites, little information is available on the comparative suitability of these procedures in procuring clean preparations of spores for experimental and morphological studies. The purpose of this study was to evaluate four techniques that had been applied previously to microsporidan and myxosporidan spores, for the isolation of Pleistophora ovariae spores from naturally infected golden shiners (Notemigonus crysoleucas). O'Grodnick's method (1975, J. Wildl. Dis. 11: 54-57) was not included. Infected ovaries from golden shiners were collected in the spring and supplied by the Oklahoma Cooperative Fishery Research Unit (Oklahoma State University, Stillwater, Oklahoma) or Ozark Fisheries (Stoutland, Missouri). Each technique was performed twice, once on infected tissue from each location. Spores were judged viable by their characteristic refractive appearance under the microscope and by their ability to extrude the polar filament when stimulated by mechanical pressure. The first method evaluated was a mortarand-pestle grinding procedure. We strained homogenate resulting from the pulverization of infected ovaries through several layers of cheesecloth, using a sterile physiological saline rinse. Then this suspension was centrifuged at 840 g for 10 min. The pellet was resuspended in saline. Spores isolated by this method were mixed with considerable debris, including large yolk droplets and particles of ovarian tissue. Although large numbers of spores were recovered, the usefulness of the preparation was limited because of the large amount of tissue material present. The second method of spore isolation was triangulation technique adapted from Cole (1970, J. Invertebr. Pathol. 15: 193-195), which involves repeated, 5-min centrifugations of 40 g and 3 g. With centrifugation at 40 g, the higher concentration of spores was in the second tube, along with large particles of debris. With centrifugation at 3 g, spores were evenly distributed among all tubes. Debris was also present in all tubes; the larger particles were in the first two tubes and smaller particles in the last two tubes. Cole, (loc. cit.) and Fowler and Reeves (1974, J. Invertebr. Pathol. 23: 3-12) prepared clean suspensions of spores from 10 species of microsporidans-all from insect hosts-by this method. Because P. ovariae infects a vertebrate host, the nature of tissue differs from that of insects and may have inhibited the separation of spores from host cells. Centrifugation at other forces, not evaluated in the present study, may be more effective for isolation of P. ovariae spores. The third method of spore isolation was a modification of the sucrose density gradient technique described by Markiw and Wolf (1974, J. Fish. Res. Bd. Can. 31: 15-20) for Myxosoma cerebralis (Myxosporida). Because