The kappa nature of opioid binding sites in a brush border membrane (BBM) fraction from human placenta has been confirmed: these sites display considerably higher apparent affinity (KI = 1.2 nM) for the kappa selective ligand U-50488 than they do for the mu and delta selective ligands [D-Ala2, MePhe4, Glyol5] enkephalin (KI = 1.5-2 microM) and [D-Thr2, Leu5] enkephalyl-Thr (KI = 10-15 microM), respectively. The BBM fraction from human placenta was incubated either with the agonist 3H-etorphine or with the antagonist 3H-diprenorphine and subsequently solubilized with digitonin. The solubilized macromolecular radioactivity was found to behave as a homogeneous entity both in molecular exclusion chromatography (app. rs = 6.1 nm) and in linear sucrose gradients (app. S20.w = 12 S). Two lines of evidence indicated that the placental kappa opioid receptor is capable of interacting with a guanine nucleotide regulatory (G) protein: (i) equilibrium binding of the agonist 3H-etorphine in the BBM fraction was clearly inhibited by 5'-guanylylimidodiphosphate (Gpp(NH)p), especially in the presence of Na+ ions while binding of the antagonist 3H-diprenorphine was significantly less so and (ii) the sedimentation velocity of the kappa opioid receptor was decreased down to about 10 S when the BBM fraction was prelabeled with radioligand in the presence of Gpp(NH)p prior to its solubilization with digitonin. The G protein that mediates the effect of Gpp(NH)p might be neither Gs nor Gi since no adenylate cyclase activity could be demonstrated in the BBM fraction from human placenta.