Neuroendocrine tissues in teleost fish aromatize androgen to estrogen at extraordinarily high rates. As part of a project in which we are studying the dynamics of sex steroid uptake, metabolism, and receptor binding in goldfish (Carassius auratus) brain and pituitary, we have identified and characterized a sex-steroid-binding component of serum. This protein has been designated a testosterone-estradiol-binding globulin (TEBG) since it bound testosterone (T) and estradiol-17 beta (E2) with high affinity (Kd = 1.9 and 2.1 nM, respectively) whereas other steroids were less effective ligands (5 alpha-dihydrotestosterone greater than progesterone = 11-ketotestosterone greater than estrone = estriol = diethylstilbestrol greater than cortisol). Scatchard analysis, disc gel electrophoresis and sucrose gradient centrifugation all indicate that T and E2 are bound by the same protein. The number of available serum binding sites (Bmax = 10(-7) M) greatly exceeded reported maximal levels of T and E2 in the same species and showed no obvious sex or seasonal differences. However, the steroid-TEBG interaction was unstable, exhibiting very short half-times of association (less than 15 min) and dissociation (less than 10 min). On the basis of comparison of the physicochemical characteristics of TEBG with other intracellular androgen-binding proteins in goldfish brain (androgen receptor, aromatase), we predict that the serum-binding protein would not limit but rather enhance exchange of T and E2 in central tissues.