In the CNS, histamine is a neurotransmitter that is inactivated by histamine N-methyltransferase (HNMT), a soluble enzyme localized to the cytosol of neurons and endothelial cells. However, it has not been established how extracellular histamine, a charged molecule at physiological pH, reaches intracellular HNMT. Present studies investigated two potential routes of histamine inactivation in mouse brain nerve terminal fractions (synaptosomes): (i) histamine uptake and (ii) histamine metabolism by HNMT. Intact synaptosomes demonstrated a weak temperature-dependent histamine uptake (0.098 pmol/min-mg protein), but contained a much greater capacity to metabolize histamine by HNMT (1.4 pmol/min-mg protein). Determination of the distribution of HNMT within synaptosomes revealed that synaptosomal membranes (devoid of soluble HNMT) contribute HNMT activity equivalent to intact synaptosomes (14.3 ± 2.2 and 18.2 ± 4.3 pmol/min-tube, respectively) and suggested that histamine-methylating activity is associated with the membrane fraction. Additional experimental findings that support this hypothesis include: (i) the histamine metabolite tele-methylhistamine (tMH) was found exclusively in the supernatant fraction following an HNMT assay with intact synaptosomes; (ii) the membrane-bound HNMT activity was shown to increase 6.5-fold upon the solubilization of the membranes with 0.1% Triton X-100; and (iii) HNMT activity from the S2 fraction, ruptured synaptosomes, and synaptosomal membranes displayed different stability profiles when stored over 23 days at − 20°C. Taken together, these studies demonstrate functional evidence for the existence of membrane-bound HNMT. Although molecular studies have not yet identified the nature of this activity, the present work suggests that levels of biologically active histamine may be controlled by an extracellular process.