ABSTRACT: Studies that screen for metabolites produced in ruminants are actively underway. We aimed to evaluate the metabolic profiles of five biofluids (ruminal fluid, serum, milk, urine, and feces) in dairy cow by using proton nuclear magnetic resonance (1H-NMR) and provide a list of metabolites in each biofluid for the benefit of future research. We analyzed the metabolites in five biofluids from lactating cows using proton nuclear magnetic resonance imaging; 96, 73, 88, 118, and 128 metabolites were identified in the five biofluids, respectively. In addition, 8, 6, 9, and 17 metabolites were unique to ruminal fluid, serum, milk, and urine, respectively. The metabolites present at high concentrations were: acetate, propionate, and butyrate in ruminal fluid; lactate, glucose, and acetate in serum;