In vitro quantitative autoradiography was used to determine the distribution of [3H]U-69593 binding sites in rat brain. The highest density of binding was found in areas of the tel- and diencephalon while certain areas of the mes- and metencephalon were moderately labeled. The distribution of [3H]U-69593 binding sites corresponds closely (but not precisely) to the distribution of sites labeled by [3H]EKC and [3H]bremazocine at room temperature but differs substantially from the distribution of sites labeled by [3H]EKC at 4 degrees C (as described by others). These anatomical findings support previous biochemical evidence indicating that [3H]U-69593 selectively labels a kappa opioid receptor subtype with characteristics that differ from the kappa receptors labeled by [3H]EKC at 4 degrees C.