We have applied a competitive protein binding technique to the assay of aldosterone, utilizing the aldosterone binding protein (ABP) in kidney as the specific receptor molecule. Intracellular ABP was obtained from the homogenization of adrenalectomized rat kidneys in 0.25 M sucrose. The 10 000 × g supernatant contained the greatest binding affinity for aldosterone and was used without further modification. 1,2-3H-aldosterone was used as the tracer. After maximum binding, which occurred after 5 hours of incubation at 0–4°C, the aldosterone-ABP complex was stable for several hours and could be separated from unbound aldosterone by gel filtration using a 1 × 15 cm Biogel P-10® column. However, since the aldosterone-ABP complex was relatively easily dissociated by warm temperatures, all procedures had to be conducted at 4°C. Competitive inhibition of aldosterone binding to ABP occurred with 9-alpha fluorocortisol, cortisol, and progesterone. No inhibition of binding was found with oestradiol. Using this in vitro binding system, 1.05 × 10−12 and 2.10 × 10–12 mol aldosterone were assayed with coefficients of variation of 13.4% and 13.3%, respectively. Because cortisol effectively competes with aldosterone for ABP, application of this technique to biological fluids must be preceded by separation of aldosterone from cortisol. However, when this separation was achieved with one-step descending paper chromatography, our blank value was high and variable. This is apparently because some material which eluted from the paper interfered with the association of aldosterone and ABP. Therefore, this sensitive assay for aldosterone cannot be applied to biological specimens until the problem of the non-specific blank is circumvented.