We have studied the concentration and properties of a protein which binds coitisol in human milk in samples obtained from women during the first 100 days after delivery. A filter disk assay was developed both for the measurement of plasma corticosteroid-binding globulin (CBG) and for the cortisol-binding protein in milk. The concentration of CBG in milk, expressed as its capacity to bind coitisol, is highest on the day of delivery, ca. 0.80 μg/dl, falls over the next 10 days to ca. 0.25 μg/dl, and remains at that level thereafter. If the concentration of CBG is expressed relative to the concentration of serum albumin in milk, it increases from day 1 to day 3 and then remains constant. A detailed comparison of CBG derived from milk and plasma showed that the two proteins co-migrated on Sephadex, sucrose gradient ultiacentrifugation, and polyacrylamide gel electrophoresis. The two proteins had the same affinity for coitisol, progesterone, 17-OH-progesterone, and dexamethasone. Furthermore, the binding activity of CBG in milk was neutralized with anti-CBG antibodies raised against CBG isolated from plasma. Unlike CBG, the concentration of coitisol in milk, 0.8–3.5 μg/dl, showed no systematic variation as a function of the postpaitum day on which the sample was obtained.