A significant decrease in the number of 3H-quinuclidinyl benzilate (3H-QNB) binding sites in striatal membranes was observed in rats exposed to gamma-butyrolactone (GBL) during 3 weeks. This decrease in 3H-QNB binding was not due to a direct effect of GBL on muscarine receptors since no alterations of binding were produced by high concentrations of GBL or of its active metabolite gammahydroxybutyric acid (GHB) when added in vitro. Challenge doses of pilocarpine were less effective in producing catalepsy and tremors in rats chronically treated with GBL. It is proposed that the partial inhibition of the nigro-striatal dopaminergic pathway produced by chronic GBL treatment leads to a persistent activation of the intrastriatal cholinergic interneurons, which are tonically inhibited by dopaminergic nigrostriatal neurons. The activation of striatal cholinergic interneurons results in a decreased number of muscarine receptors and in a lower sensitivity to cholinomimetic agents. Moreover, tolerance to the anesthetic effect of GBL was found to occur after chronic treatment with this drug. The brain levels of GHB at the time of regaining of the righting reflex were 50% higher in GBL pretreated animals then in controls. Indicating the tolerance to GBL-induced "sleep" is due to a decrease sensitivity of the effector to GHB rather than to a faster inactivation of elimination of the drug.