The purpose of this study was to investigate the olfactory transfer of a growth hormone releasing neuropeptide, hexarelin to the brain tissues by comparing brain uptake levels after intranasal administration with those after intravenous administration. The hexarelin nasal formulation was prepared using an aqueous cosolvent vehicle consisting of ethanol, propylene glycol, and n-tridecyl-beta-D-maltoside as a permeation enhancer. Hexarelin was administered intravenously or intranasally to male rabbits at a dose of 1 mg/kg. Drug concentrations in the plasma, cerebrospinal fluid and six different regions of the brain, i.e., olfactory bulb (OB), olfactory tract (OT), anterior (CB1), middle (CB2), posterior (CB3) cerebrum, and cerebellum (CL) were analyzed by LC/MS method after solid phase extraction. The brain and cerebrospinal fluid levels achieved following intranasal administration were approximately 1.6 times greater than those attained after intravenous administration despite the intranasal plasma levels being significantly lower than the intravenous plasma levels. Intranasal administration resulted in significantly different spatial distribution patterns in various regions of brain with the rank order of C(OB)>C(OT)>C(CB1, CB2, CB3)>C(CL) at 10, 20, and 40 min post-dosing, whereas intravenous administration yielded nearly similar distribution patterns in the brain. The intranasal administration into one nostril (left or right) exhibited markedly greater hexarelin concentrations in olfactory bulb and olfactory tract on the treated-side of brain tissues than those on the non-treated-side of the brain hemisphere. It was demonstrated that the hydrophilic neuropeptide hexarelin was transferred via olfactory pathway to the brain hemispheres and the drug transfer via this route significantly contributed to high brain concentrations after nasal administration to rabbits.