The histamine H3 receptor (H3R) modulates the release of neurotransmitters that are involved in vigilance, cognition, and sleep-wake regulation. H3R antagonism has been proposed as a novel approach to the treatment of cognitive and attention deficit as well as sleep disorders. It is apparent that H3R antagonists produce pharmacological effects in preclinical animal models across a wide dose range. Several H3R antagonists were reported to be effective at producing cognitive enhancing effects at low doses, while producing robust wake enhancement at higher doses. To better understand the effect of H3R antagonists across a broad dose range, an ex vivo receptor binding assay has been used to estimate the degree of H3R occupancy in vivo. The H3R antagonists ciproxifan, thioperamide, GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride), and ABT-239 ([4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile) produced wake-promoting activity in vivo and a dose-dependent inhibition of H3R binding ex vivo. For ciproxifan, thioperamide, and GSK189254, a relatively low level of cumulative wake activity was linearly correlated with up to 80% of the receptor occupancy. In contrast, an abrupt break from linearity and a robust increase of waking activity was observed at doses that produce greater than 80% occupancy. Our results suggest a relatively small increase of waking activity at low levels of receptor occupancy that may be consistent with reported enhancement of attention and cognitive function. Robust waking activity at higher levels of H3R occupancy may be mechanistically different from activities at low levels of H3R occupancy.