Walleye (Stizostedion vitreum) is a species of interest for the diversification of North American aquaculture production, and semen cryopreservation is of particular value to this effort. To test the hypothesis that adjusting semen extender composition and dilution ratio increases sperm quality after thawing, three extenders (Ext1, Ext2, Ext3; all with DMSO as a cryoprotectant) and three dilution ratios (semen/extender: 1:5, 1:9, 1:15) were screened. The best results were obtained when semen was diluted at a 1:15 ratio with Ext 1, Rathbun extender supplemented with 7% DMSO, 4 mg/ml BSA and 7.5 mg/ml ProFam, a soy-based protein (P = 0.05, n = 6). This method resulted in 46 +/- 3% motility of the thawed spermatozoa and a mortality rate of 39 +/- 4% whereas Ext2 and Ext3 resulted in motility rates of only 10 and 5%. respectively. To test an additional hypothesis that phosphodiesterase inhibition improves sperm function, we assessed the fertility of sperm frozen in optimal conditions and thawed in the presence or absence of 5 mM theophylline (n = 5). The best result was achieved in water without theophylline, with fertilization rates ranging from 28.51 +/- 6.84 to 59.02 +/- 1.06% eyed-up stage, and theophylline reduced fertility (P < 0.05). Our data show that Ext1 at a dilution ratio of one part semen to 15 parts extender should be used for walleye semen cryopreservation and that the fertilizing media does not benefit from theophylline supplementation.