Cortisol binding by dog plasma was studied by ultrafiltration and equilibrium dialysis techniques and was compared to human plasma using 3 criteria: 1) the binding curve; 2) the sensitivity of the binding proteins to heat inactivation; and 3) the effect of estrogen administration on the cortisol-binding properties of the plasma. The resting plasma cortisol level in the dog is below 2 μg/ 100 ml, and at this level, by ultrafiltration analysis, about 88% of the cortisol is bound. At a plasma level of 10 μg/100 ml, 67% of the cortisol is bound and at 100 μg/100 ml, 56% is bound. A 4% dog albumin solution binds about 50% of the cortisol. Human transcortin is inactivated by heating the plasma at 60 C for 20 min; dog plasma shows little change in binding characteristics following this period of heating. Human transcortin increases in concentration during estrogen administration. Estrogen treatment of 4 dogs did not change their plasma cortisol-binding properties. Dog plasma, therefore, contains a protein (s) similar to human transcortin, with high affinity for cortisol. The concentration of this protein in dog plasma, however, provides much less cortisol-binding capacity than does transcortin, and, unlike transcortin, this protein does not increase in concentration with estrogen treatment and does not show similar heat lability.