Abstract To elucidate whether the behavioural action of cortisol 21-sulphate (F-21-S) on the central nervous system (CNS) can be explained on the basis of steroid-receptor binding, the effect of F-21-S on cortico-sterone (B) binding to cytosol protein was investigated. Rat brain cytosol was prepared essentially according to the method of Roth. Approximately 1.25 mg of cytosol protein was incubated with 1 pmol (10 5 d.p.m.) of [ 3H]-B in 500 μl of assay mixture at 0°C for 4 h with and without cold B or cold F-21-S. Dissociation constant ( K D ) and binding maximum ( B max) of B-rat brain cytosol receptor protein(s) was 4.0 × 10 i−9 M and 2.9 × 10 −13 mol per mg cytosol protein, respectively. F-21-S did not exhibit on this binding as lower concentration than 200 nM. The results suggest mechanism(s) other than steroid receptor binding for the behavioural action of F-21-S.