In this article an algorithm estimating the basic metabolic fluxes in lactating cows is described that uses data of substrates absorption, heat production, respiratory quotient and milk productivity. The flux diagram permits to estimate parameters which are very difficult to measure in physiological experiments, including rates of oxidation of substrates to CO₂ and their interconversions (synthesis of long-chain fatty acids from acetate, gluconeogenesis from amino acids, etc.). The developed model was verified in an experiment carried out on two groups of lactating cows with measurements of energy balance, heat production and respiratory quotient (СО₂ /О₂) during the periods of 40-45, 60-65 and 90-95 days of lactation. The estimates of oxidation rates obtained for amino acids, glucose, long chain fatty acids and acetate do agree with literature data. The analysis of combined data revealed a negative correlation between oxidation rates of glucose and acetate (r=0.91) and between substrate groups (long chain fatty acids + butyrate) and (glucose + acetate) (r=0.75).