SummaryThis study deals with the determination of 2,3‐DPG concentrations in canine blood. The results obtained demonstrate the intraerythrocytic location of this organic phosphate as well as its equimolar relationship with the haemoglobin tetramers in blood obtained from clinically healthy dogs. The average value obtained for this group of healthy dogs (n = 93) was 5.81 ± 0.07 mmol of 2,3‐DPG/L of erythrocytes. In dogs with cardio‐pulmonary insufficiency (n = 12) and uraemic syndrome (n = 10), a greater concentration of 2,3‐DPG was observed, together with a loss of the equimolar relationship between this phosphate and the Hb tetramers. Furthermore, in the group of dogs with uraemic syndrome we observed a highly significant correlation between the blood values of urea and the concentration of 2,3‐DPG expressed in relation to the haemoglobin