Hydroxyurea (HU) is an anti-proliferative and anti-microbial compound that is widely used in medical therapy but is also found to occur naturally in different phylogenetic groups. However, it is still unknown how this compound is endogenously synthesized. I hypothesized that HU is synthesized through either the diversion of hydroxyarginine from the nitric oxide synthase (NOS) pathway by arginase, or the hydroxylation of urea by cytochrome P450. To test the possible pathways involved in HU synthesis, I incubated isolated Leucoraja erinacea mitochondria with stable isotope labelled arginine and enzymatic inhibitors and determined the activity of mitochondrial arginase and NOS. While HU was found to be mainly mitochondrial, the inhibition of either enzyme had no effect on the rate of HU production, suggesting that another pathway is involved in HU synthesis. The pathway for HU synthesis is still unknown. However, this study contributes to the known evolutionary timeline for mitochondrial NOS.