The efficient import of choline into cholinergic nerve terminals by the presynaptic, high-affinity choline transporter (CHT, SLC5A7) dictates the capacity for acetylcholine (ACh) synthesis and release. Tissue levels of ACh are significantly reduced in mice heterozygous for a loss of function mutation in Slc5a7 (HET, CHT+/-), but significantly elevated in overexpressing, Slc5a7 BAC-transgenic mice (BAC). Since the readily-releasable pool of ACh is thought to constitute a small fraction of the total ACh pool, these genotype-dependent changes raised the question as to whether CHT expression or activity might preferentially influence the size of reserve pool ACh vesicles. In the current study, we approached this question by evaluating CHT genotype effects on the release of ACh from suprafused mouse forebrain slices. We treated slices from HET, BAC or wildtype (WT) controls with elevated K+ and monitored release of both newly synthesized and storage pools of ACh. Newly synthesized ACh produced following uptake of [3H]choline was quantified by scintillation spectrometry whereas release of endogenous ACh storage pools was quantified by an HPLC-MS approach, from the same samples. Whereas endogenous ACh release scaled with CHT gene dosage, preloaded [3H]ACh release displayed no significant genotype dependence. Our findings suggest that CHT protein levels preferentially impact the capacity for ACh release afforded by mobilization of reserve pool vesicles.