Objective: To examine differences in the metabolite, steroid and lipopolysaccharide of follicular fluid collected from the ovulatory follicle of Holstein repeat breeding cows, lactating cows, and virgin fertile heifers. Methods: Estrus was induced in animals possessing functional corpus luteum by intramuscular administration of prostaglandin F2 α. Six to twelve hours after detection of the standing estrus, cervical samples were collected to detect subclinical endometritis via counting neutrophils on stained smears of cervical swabs. Then, follicular fluid of ovulatory follicles and serum samples were collected from repeat breeding cows (n=11), lactating cows (n=8) and virgin fertile heifers (n=10). Sodium and potassium were measured with a flame photometer method. Urea, total protein, glucose, cholesterol and β -hydroxybutyric acid were assayed with commercial spectrophotometry kits. Chloride concentration was also measured with titration of samples against silver nitrate. Progesterone, estradiol-17 β and lipopolysaccharide concentrations were measured using enzyme-linked immuno sorbent assay kits. Results: All analysis of follicular fluid samples showed that repeat breeding and lactating cows had a mean higher lipopolysaccharide concentration than that of the virgin fertile heifers (P Conclusions: High follicular fluid concentration of lipopolysaccharide in ovulatory follicles results in the occurrence of repeat breeding syndrome in dairy cows. Further, a lower serum estradiol-17 β concentration and a higher percentage of neutrophil in the cervical secretion on the day of artificial insemination may have resulted in the occurrence of repeat breeding syndrome in dairy cows.