Pure separation of various leukocytes is required for the assessment of their roles in immunological and physiological function. In this study, pure separation of monocytes from canine peripheral blood was attempted. At first, mononuclear cells (PBMC) were separated by ficoll-hypaque gradient method and then monocytes were recovered from PBMC suspensions in sucrose gradient Sol. (PBMC-Sucrose), autologous plasma (PBMC-Plasma) and autologous serum (PBMC-Serum) incubated at 37deg C for 2 hours. In the separation of PBMC by ficoll-hypaque gradient method in canine blood, higher relative centrifugal force (RCF) was required, as high as more than 1,300xg RCF for 40 minutes, for clear formation of PBMC layer than that in human blood as usually used 400xg RCF for 40 minutes. In monocytes-separation from three PBMC suspensions following PBMC separation, recovery-, purity- and viability- rate of monocytes showed better results in PBMC-Plasma and PBMC-Serum than in PBMC-Sucrose suspension, particularly showing better results from PBMC suspensions performed by centrifugation at 1,500xg RCF for 40 minutes