High affinity binding of cortisol, progesterone, testosterone, and estradiol by plasma proteins was examined in 29 marsupial species by equilibrium dialysis and/or polyacrylamide gel electrophoresis. The association constant (Ka, 36°) and binding capacity ([Σρ{variant}]) for cortisol and testosterone were measured by microdialysis and found to be 3.2 to 9.6 × 107 M-1 and 0.3 to 3.7 × 10-7 M respectively, for cortisol and 1.6 to 8.3 × 108 M-1 and 0.3 - 4.0 × 10-7 M for testosterone. High-affinity binding of cortisol was detected in all 25 species studied by this method but testosterone was bound in 9 only. Steroid-binding proteins were further characterized by polyacrylamide gel electrophoresis of plasma equilibrated with radioactive steroid at 4°. Two types of non-albumin binding proteins were identified, one with a mobility like human corticosteroid-binding globulin (CBG) and an affinity for both cortisol and progesterone and a second protein with a mobility like human sex hormone-binding globulin (SHBG) and an affinity for testosterone. High affinity binding of estradiol was not detected, and a second CBG was found in four species. © 1979.