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The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC–CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [3H]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC–CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC–CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC–CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC–CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC–CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity.