The characteristics of specific binding of the ATP-sensitive K(+) (K(ATP)) channel blocker [3H]glibenclamide to forebrain membranes (P(2) fraction, 4 degrees C) obtained from morphine-naive and -tolerant mice were evaluated. Morphine tolerance was induced by osmotic minipumps that released 45 mg/kg/day of morphine subcutaneously for 6 days. This treatment enhanced the antinociceptive ED(50) of morphine without changing its E(max). In morphine-naive animals, (1) both the association and the dissociation of [3H]glibenclamide were biphasic; (2) [3H]glibenclamide was displaced by other sulfonylureas (order of potency: glibenclamide>glipizide&z.Gt;tolbutamide) with pseudo-Hill coefficients lower than unity and biphasic Hofstee plots; and (3) Scatchard plots of saturation experiments were curvilinear, showed a Hill coefficient of 0.81+/-0.04 and suggested the presence of two binding sites with a K(D) of 0.13 and 3.17 nM and a B(max) of 12.30 and 84.47 fmol/mg protein, respectively. By contrast, in membranes obtained from morphine-tolerant animals, (1) the Scatchard plots showed only one population of binding sites with a K(D) of 0.87 nM and a B(max) of 77.99 fmol/mg protein, and the Hill coefficient was very close to unity (0.96+/-0.1); (2) competition experiments (using glibenclamide as displacer) showed a pseudo-Hill coefficient of 0.99+/-0.04; and (3) dissociation experiments showed only one phase of dissociation. These results suggest that [3H]glibenclamide binds to two different sites in membranes obtained from morphine-naive animals, but to only one site in morphine-tolerant animals. Consequently, it seems that morphine tolerance in mice involves adaptive changes in K(ATP) channels.