Choline acetyltransferase (ChAT) is the key-enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay but can only be performed on postmortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real-time using nuclear magnetic resonance (NMR) spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted in order to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used (15) N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before (1) H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques. © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/jnc.12025.