PurposeTo detect local cholinergic changes in human medial temporal lobe during configural working memory performance.Materials and MethodsProton magnetic resonance spectroscopy (1H‐MRS) measurements were acquired at 3T from a 2 × 2 × 3 cm voxel in right medial temporal lobe from 36 subjects during performance of a configural visual working memory task (cWMT). In order to compensate for expected task‐based blood oxygenation level‐dependent (BOLD) T2* effects, resonance signal changes of unbound choline‐containing metabolites (Cho) were referenced to an internal standard of creatine + phosphocreatine metabolites (Cre) and compared between four task blocks: rest, memorization, active memory maintenance, and recognition. An unannounced memory retention test was conducted in 21 subjects. Quality assurance analyses examined task‐based Cho and Cre individually as well as referenced to resonance signal from N‐acetylaspartate (NAA).ResultsIncreases from a resting baseline in the Cho/Cre ratio were observed during 60‐second blocks of active memory maintenance across the group (P = 0.0042). Behavioral accuracy during task performance correlated with memory retention (r = 0.48, P = 0.027). Quality assurance measures showed task‐based changes in Cre resonance signal both individually (P = 0.00099) and when utilized as a noncholinergic internal reference (NAA/Cre, P = 0.00079).ConclusionIncreases in human medial temporal lobe 1H‐MRS Cho/Cre ratio occur during the maintenance of configural working memory information. However, interpretation of these results as driven by cholinergic activity cannot be assumed, as NAA, a noncholinergic metabolite, shows similar results when utilizing Cre as a reference. Caution is advised when considering Cre as an internal standard for task‐based 1H‐MRS measurements.Level of Evidence: 2Technical Efficacy: Stage 1J. Magn. Reson. Imaging 2018;47:682–691.