The biosynthesis of L-carnitine in eukaryotic organisms was first elucidated in the ascomycete Neurospora crassa. The first step of the pathway is catalysed by epsilon-N-trimethyllysine hydroxylase (TMLH), which converts epsilon-N-trimethyllysine into beta-hydroxy-N-epsilon-trimethyllysine in a reaction dependent on alpha-ketoglutarate, Fe2+ and oxygen. Here we report on the cloning of the N. crassa TMLH cDNA and its functional expression in Saccharomyces cerevisiae. The TMLH cDNA contains an open reading frame of 1413 base pairs encoding a predicted polypeptide of 471 amino acids. The Michaelis-Menten constants of the heterologously expressed enzyme were determined for epsilon-N-trimethyllysine, alpha-ketoglutarate, Fe2+ and correspond to 0.33 mM, 133 microM and 46 microM, respectively.