As lactating cows in severe negative energy balance have poor reproductive performance, the effect of dietary fat supplementation (fish oil, soybean oil) on PGFM secretion, ovarian function and blood metabolites is investigated. In this experiment, the effects of dietary polyunsaturated fatty acids on plasma metabolites, ovarian function and prostaglandin secretion of 20 primiparous Holstein cows was studied. The cows were randomly allocated to one of four groups that were fed either: 1) a control diet; 2) a diet with 3% (Feed dry matter basis) fish oil; 3) a diet with 3% soybean oil; or 4) a diet with 1.5% fish oil and 1.5% soybean oil. Groups were synchronized using the heat-synch method and were fed their respective diets for 35 days, allowing 14 days for dietary adaptation and 21 days for data collection. Concentration of plasma glucose, triglycerides and low density lipoprotein (LDL) cholesterol were not affected by the treatments, but plasma total cholesterol and high density lipoprotein (HDL) cholesterol concentrations were significantly higher in cows that consumed the oil-containing diets (p < 0.05).The number of follicles, corpus luteum size and plasma estradiol, progesterone and prostaglandin F2? metabolite (PGFM) concentrations were similar across all treatments. However, the size of the largest follicle was significantly greater in cows that consumed a diet containing fish oil or soybean oil (p < 0.05). These results suggest that polyunsaturated fatty acids can influence both ovarian and uterine function in cows, but further studies are required to test their effects on dairy cow reproduction.