Trimethyllysine hydroxylase (TMLH) catalyses C-3 hydroxylation of Ne-trimethyllysine in the first step of carnitine biosynthesis in humans. Studies on TMLH have been hampered by the lack of established chemical methods. We report that an Ne-trimethyllysine analogue that contains the fluoromethyl group can be used as a H-1 and F-19 NMR probe for studies on TMLH catalysis.