[3H]U-69593 is an opiate agonist that has been reported to bind in vitro with high affinity and selectivity to the kappa receptor subtype. The studies reported here were designed to determine the optimal conditions for labeling kappa receptors with [3H]U-69593 and to further characterize the binding site. The effects of temperature and NaCl on [3H]U-69593 binding were of particular interest because previous studies reported that [3H]ethylketocyclazocine ([3H]EKC) and [3H]bremazocine binding to kappa receptors was optimal at 4 degrees C in the presence of NaCl. Those conditions were not found to be optimal for [3H]U-69593 binding. Although the pharmacological specificity and Bmax of [3H]U-69593 binding was similar at room temperature and at 4 degrees C, the binding affinity was approximately three times lower at 4 degrees C than at room temperature. In addition, NaCl had an effect on [3H]U-69593 binding that was opposite that on [3H]EKC binding at 4 degrees C (100 nM DAGO and 100 nM DADLE were included in all [3H]EKC assays to prevent binding to mu and delta receptors), i.e. NaCl decreased, rather than increased, [3H]U-69593 binding at 4 degrees C. These differences between [3H]U-69593 and [3H]EKC binding at 4 degrees C were accentuated by a vast difference in the density of the binding sites [Bmax approximately equal to 12 fmol/mg protein for [3H]U-69593 vs approximately equal to 375 fmol/mg protein for [3H]EKC at 4 degrees C in the presence of NaCl) and suggested that [3H]U-69593 might bind selectively to a kappa receptor subtype. This concept was supported by competition experiments. In particular, the site labeled by [3H]EKC at 4 degrees C was found to be relatively insensitive (compared to [3H]U-69593 and [3H]EKC binding at room temperature) to the kappa agonist U-50488H, a close analog to U-69593. Based on these findings, we propose that [3H]U-69593 (and U-50488H) labels a kappa receptor subtype which differs from that labeled by [3H]EKC at 4 degrees C.