The purpose of this study was to examine the influence of the corpus luteum on hormonal and biochemical metabolites composition of follicular fluid (FF) harvested from different sized follicles and their relationship to blood serum in dairy cows. Ovaries were recovered from 30 female adult cows (Holstein Friesian) 4-7 years of age with clinically normal reproductive tracts after slaughtering. Blood samples were collected from the jugular vein before slaughter from each cow. The stage of the cycle in the cows slaughtered was diestrus determined post mortem. The ovaries collected per cow were classified with corpus luteum (CL+) and without corpus luteum (CL-). Visible follicles on the surface of the ovaries were classified, based on their diameter, into (i) small (3-5 mm), (ii) medium (6-9 mm) and (iii) large (10-20 mm) categories. Follicular fluid was aspirated from different sized follicles in CL+ and CL- ovaries. Serum and FF samples were analyzed for hormones (estradiol-17β, progesterone, testosterone, T3 and T4) and biochemical metabolites (glucose, cholesterol, triglyceride, total protein, albumin and globulin). Results showed that the FF concentration of estradiol-17β, progesterone