This study describes an exchange assay for measurement of cytosolic and nuclear androgen receptors (AR) in human and dog prostates. Efficient replacement of endogenously bound ligand from the receptor with [3H] mibolerone was achieved by incubation of cytosolic or nuclear fractions at 0 degree C for 72 h in the presence of 0.15 M NaSCN and 15% sucrose. It was demonstrated that in the presence of the chaotropic salt rapid steroid dissociation took place at 0 degree C followed by [3H] mibolerone binding; sucrose protected the AR from denaturation by NaSCN. Combination of these two reagents, therefore, allowed quantitative androgen exchange at 0 degrees C. Receptors determined by this exchange procedure are specific for androgens, of high affinity (KD 2-5 nM), and sedimented on sucrose gradients as 4-4.6 S entities. Use of [3H] mibolerone minimized interference from plasma proteins and reduced nonspecific binding. This exchange assay has now been applied to quantitation of cytosolic and nuclear AR in small tissue samples (50 mg). Thus it is possible to measure AR in tissue samples obtained by needle biopsies and attempt to correlate receptor values to clinical response of prostatic cancer patients.