The last step in L-carnitine biosynthesis in eukaryotic organisms is mediated by ${gamma}$-butyrobetaine hydroxylase (EC1.14.11.1), a dioxygenase that converts ${gamma}$-butyrobetaine to L-carnitine. This enzyme was previously identified from rat liver and humans, and the peptide sequence of human ${gamma}$-butyrobetaine hydroxylase was used to search the Neurospora crassa genome database, which led to an identification of an open reading frame (ORF) consisting of 1,407 bp encoding a polypeptide of 468 amino acids. When this protein was expressed in Saccharomyces cerevisiae, the crude cell-free extract exhibited ${gamma}$-butyrobetaine hydroxylase activity.