In in vivo 1H spectroscopy, the signal at 1.32 ppm is usually assigned to lactate. This resonance position is shared with threonine at physiological pH. The similarity of spectral patterns of lactate and threonine renders the separate measurement of either threonine or lactate without and even with editing technically challenging. In this study, the threonine signal was detected using a single-shot multiple-bond editing technique and quantified in vivo in both rat and human brains. A threonine concentration was estimated at 0.8 ± 0.3 mM (mean ± SD, n = 6) in the rat brain and at ∼0.33 mM in the human brain.