This study was conducted to evaluate whether "Royal jelly" (RJ) added to Tris-buffer dilute contributed to supporting post-thaw viability and longevity of frozen canine spermatozoa. Two Japanese spitzs (2 to 4 years of age) were used as a semen donor. Semen was collected by manual masturbation and separated into 3 fractions. Only the sperm-rich fraction having sperm motility of more than 70%, containing sperm concentration of 2~4$ imes$10$^{8}$ cells/ml and having dead or abnormal spermatozoa of less than 15% was used for the experiment. Each ejaculated semen was centrifuged at 400 $ imes$ g for 5 min and then diluted in a Tris-buffer supplemented with 20 ml egg yolk (Ext I), 4% glycero1 and 1% Equex STM Paste (Ext II) or g1ycero1, Equex STM paste and RJ of various concentrations (Ext II-RJ). After freezing and thawing, viability of spermatozoa in Ext II -RJ containing 1% RJ immediately after thawing (67.5$pm$9.6) was significantly lower than that of Ext II , Ext II -RJ containing 0.01 or 0.1% RJ (77.5$pm$12.5, 78.7$pm$8.2 and 80.0$pm$6.3). However, Ext II-RJ containing 0.1% RJ yielded higher viability than Ext II, Ext II-RJ containing 0.01% at or 1% 1 h after thawing (69.5$pm$8.1 vs. 55.0$pm$12.9, 57.5$pm$9.6 and 41.5$pm$12.6; P<0.05). At 1 h after thawing, the viability of spermatozoa thawed in 7$0^{circ}C$ (68.8$pm$12.5) was significantly higher than that of spermatozoa thawed in 38$^{circ}C$ (48.8$pm$16.3), although there was no difference in the viability between both groups immediately after thawing (77.5$pm$9.6 and 81.3$pm$8.1). Post-thaw viability and longevity of post-thaw spermatozoa in Ext II-RJ containing 0.1% RJ was higher in those in Ext II at 1 h (65.0$pm$12.9 vs. 42.5$pm$12.6), 2 h (52.5$pm$12.6 vs. 27.5$pm$17.1) and 3 h (40.0$pm$14.1 vs. 20.0$pm$12.1) after thawing. These results indicated that addition of 0.1% af to Tris-buffer enhanced post-thaw viability and longevity of canine spermatozoa and this additive can be used for increasing the possibility of collision between spermatozoa and ova during insemination.emination.