Dietary unsaturated fatty acids (UFAs) have been implicated in several reproductive processes in dairy cows through a variety of mechanisms. This study examined the effects of periparturient supplementation of rumen bypass fats low or high in proportion of UFAs (oleic and linoleic) on preovulatory follicle characteristics. Forty-two 256-day pregnant dairy cows were divided into three groups and were fed a control diet (n= 14) or supplemented with fats either low (LUFA; n= 14) or high (HUFA; n= 14) in UFAs. At 14-15 days following behavior estrus, the cows received a prostaglandin F2x injection and 48 h later > 7 mm follicles were aspirated. Progesterone (P4), androstenedione (A4), and estradiol (E2) were determined in the follicular fluid. Out of 75 follicles, 37 follicles that were aspirated between 55 and 70 days post partum were regarded as E2-active follicles (E2/P4 ratio > 1) and subjected for further analysis. The diameter of preovulatory follicles was greater in cows fed HUFA than in those fed control or LUFA. The concentrations and content of A4 and E2 in follicles and E2/P4 ratio were higher in the HUFA group than in the other two groups. The P450 aromatase mRNA expression in granulosa cells that were collected from the aspirated preovulatory follicles was also higher in the HUFA cows than in the other groups. A significant correlation was observed between E2 concentrations in preovulatory follicles and E2 concentrations in plasma at aspiration. In conclusion, dietary UFA increased the size of and elevated steroid hormones in preovulatory follicles, which may be beneficial to consequent ovarian function.