The binding in vitro of an opiate agonist, (³H)-morphine, was studied using rat brain slices which were incubated in the modified Krebs-Henseleit bicarbonate buffer solution containing various concentrations of electrolytes with or without morphine, naloxone or morphine+naloxone at 4˚C for 24 hours. The binding of (³H)-morphine may be seperated into two component; one a saturable binding and the other nonsaturable. The saturable binding may be calculated from the differences in binding observed in the absence and presence of high concentration of morphine. The maximal saturable binding and K_D value in the naive preparations were 0.32 ± 0.02 pmole/mg protein and 0.75 ± 0.07 nM respectively. The saturable binding of (³H)-morphine was significantly increased by low temperature-treatment, while K_D value was not changed. Morphine in the incubation media significantly increased the saturable binding of (³H)-morphine and K_D value. Naloxone also increased the maximal saturable binding of (³H)-morphine and K_D value of the drug. Decrease of K⁺ and Mg⁺⁺, and addition of Mn⁺⁺ in the incubation media significantly increased the saturable binding of (³H)-morphine, but decrease of Na⁺and increase of Ca⁺⁺ in the incubation media did not influence the binding. The increment of the saturable binding of (³H)-morphine by nonlabeled morphine in the incubation media was notaffected by decrease of Na⁺, K⁺ or Mg⁺⁺, or addition of Mn⁺⁺ into the incubation media, but was inhibited by increase of Ca⁺⁺ in the incubation media, while the increment of the saturable binding of (³H)-morphine was net observed by decrease of Na⁺, K⁺ or Mg⁺⁺, or increase of Ca⁺⁺ in the incubation media. The above results indicate that change of opiate binding sites in quality, i.e. affinity, and quantity, i.e. number of binding sites, may occur by low temperature-treatment in the absence and presence of morphine or naloxone and that electrolytes play role of the changes of opiate binding sites.