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Neurotensin (NT) receptor subtypes were investigated in nine brain regions from long sleep (LS) and short sleep (SS) mice that were selectively bred for differences in sensitivity to ethanol. Differences in NT receptor densities may mediate, in part, genetically selected differences in ethanol sensitivity between the two lines of mice. The use of [3H] NT at concentrations from 0.02 to 20 nM yielded biphasic binding isotherms as revealed by Scatchard analysis. Membranes from LS ventral midbrain yielded dissociation constants (KD values) of 0.34 and 3.85 nM for the high (NTH) and low (NTL) affinity components, respectively. SS membranes displayed similar KD values, however the maximum number of binding sites (Bmax) for both receptor subtypes were significantly greater in SS than in LS membranes (46.7 vs. 71.5 fmol/mg protein for NTH and 170.2 vs. 208.2 fmol/mg protein for NTL). Using levocabastine, and H1 antagonist with selectivity for NTL, characterization of NTH and NTL binding in nine brain regions was performed. In general, membranes from each brain region of SS mice had higher densities than LS for both receptor subtypes. Significant differences for the total density of receptors and NTL were found in entorhinal cortex, nucleus accumbens, hippocampus, and ventral midbrain. The only region to differ in NTH was the ventral midbrain. Competition experiments using various NT fragments to compete for NTH binding showed the C-terminal amino acids to be essential for binding. The order of potency was NT1-13 = NT8-13 greater than Neuromedin N greater than NT1-8 = NT1-11.