The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as <100㎛, <100㎛ to <110㎛, <110㎛ to <120㎛, >120㎛. Oocytes were cultured in culture medium supplemented with 10%FBS, 0.4%BSA,10% porcine follicular fluid (pFF), 10% canine serum (CS), or 10% canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes (>120㎛) was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of >110㎛ groups was significantly higher than that in <100㎛ group (p<0.05). The oocytes cultured in 10% FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in 10% FBS-, 0.4% BSA-, and 10% pFF-supplemented groups were significantly higher than those in 10% CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.