Guinea pig brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents, 2-(4-ethoxybenzyl)-1- diethylaminoethyl-5-isothiocyanatobenzimidazole.HCl (BIT) and N-phenyl-N-[1-(2-(4-isothiocyanato)phenethyl)-4- piperidinyl]-propanamide.HCl (FIT), were used in this study to test the hypothesis that guinea pig brain possesses subtypes of kappa receptors. Pretreatment of membranes with either (-)-(1S,2S)-U50,488 or the kappa selective acylating agent, (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetamide, caused a wash-resistant inhibition of kappa 1 binding sites labeled by [3H]U69,593 binding, but not kappa 2 binding sites labeled by [3H]bremazocine. Binding surface analysis of [3H]bremazocine binding resolved two binding sites, termed kappa 2 and kappa 2b, present at densities of 212 and 225 fmol/mg protein, which had low affinity for (-)-(1S,2S)-U50,488 and U69,593. The kappa 2b site had high affinity for beta-endorphin(1-31) (Kd = 5.5 nM) and [D-Ala2,D-Leu5]enkephalin (Kd = 14 nM), and lower affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (Kd = 147 nM) and [Leu5]enkephalin (Kd = 46.0 nM). Binding surface analysis of [3H]U69,593 binding also resolved two binding sites, termed kappa 1a and kappa 1b, present at densities of 6.0 and 40.0 fmol/mg protein. The kappa 1a binding site was characterized by very high affinity for alpha-neoendorphin. Quantitative autoradiographic studies demonstrated that kappa 2a and kappa 2b binding sites are heterogeneously distributed in guinea pig brain, and that the anatomical distribution of kappa 1 binding sites reported in the literature is different from that observed in this study for the kappa 2 binding sites. Viewed collectively, these data provide evidence for four kappa receptor subtypes in guinea pig brain.