Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and nonlinear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1) = 1.15 nM, Bmax(1) = 0.085 pmol/mg; KD(2) = 232 nM, Bmax(2) = 16.9 pmol/mg. For chicken cerebrum, KD(1) = 1.39 nM, Bmax(1) = 0.111 pmol/mg; KD(2) = 166 nM, Bmax(2) = 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.