The biologically important carnitine biosynthesis pathway in humans proceeds via four enzymatic steps. The first step in carnitine biosynthesis is catalyzed by trimethyllysine hydroxylase (TMLH), a non-heme Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase, which catalyzes the stereospecific hydroxylation of (2 S )- Nε-trimethyllysine to (2 S ,3 S )-3-hydroxy- Nε-trimethyllysine. Here, we report biocatalytic studies on human TMLH and its 19 variants introduced through site-directed mutagenesis. Amino acid substitutions at the sites involved in binding of the Fe(II) cofactor, 2OG cosubstrate, and (2 S )- Nε-trimethyllysine substrate provide a basic insight into the binding requirements that determine an efficient TMLH-catalyzed conversion of (2 S )- Nε-trimethyllysine to (2 S ,3 S )-3-hydroxy- Nε-trimethyllysine. This work demonstrates the importance of the recognition sites that contribute to the enzymatic activity of TMLH: the Fe(II)-binding H242-D244-H389 residues, R391-R398 involved in 2OG-binding, and several residues (D231, N334, and the aromatic cage comprised of W221, Y217 and Y234) associated with binding of (2 S )- Nε-trimethyllysine.