The binding of [ 3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a K d of 352 nM and a B max of 3.1 pmol/mg wet weight. In Ringer's saline, [ 3H]kainate bound with a B max of 1.8 pmol/mg wet weight and a K d of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [ 3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n- octyl-β- d-glucopyranoside . Approximately 30–50% of the total number of membrane-bound binding sites were recovered on solubilization. The K d of [ 3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [ 3H]kainate binding was identical and the competitive ligands had similar K i values in both membranes and solubilized extracts. In membrane preparations, [ 3H]kainate displayed a two component off-rate with k off values of0.97· min −1 and0.07· min −1; in solubilized extracts, however, only a single off-rate ( k off = 0.52 min −1) was observed. The hydrodynamic properties of n- octyl-β- d- glucopyranoside solubilized [ 3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.