The mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine.  Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Deoxyhypusine hydroxylase (DOHH) is the second of the two enzymes that catalyze the maturation of eIF5A.  Inhibitors DOHH have been shown to exert antiproliferative and antiretroviral effects.  A detailed knowledge of structure-function relationship of the enzyme will help in designing better inhibitors. Very recently, DOHH gene has been cloned in yeast and human and characterized as a HEAT-repeat–containing metalloenzyme. In this abstract, we report cloning and expression of bovine DOHH. Gene-specific primers were designed based on the bos taurus similar to HEAT-like repeat sequence (accession # XM_604893). PCR was performed using PCR ready first strand cDNA of bovine brain, the gene-specific primers, and FideliTaq^TM PCR master mix. Southern blot analysis of the amplified DNA showed hybridization with human DOHH cDNA probe. The DNA was subcloned into pGEX-2T and the target protein overexpressed. The identity of the DNA was confirmed by DNA sequencing. The overexpressed protein was active and able to convert deoxyhypusine form of eIF5A to hypusine form of eIF5A. Supported by National Institute of Health 1R15 GM 60266-01A1 and the University Research Council, Western Illinois University.