Metformin is one of the most often prescribed medications in the world with over 80 million prescriptions per year in the U.S. alone, and its use is expected to increase as the global prevalence of diabetes rises. Once taken, metformin is not metabolized in the body and finds its way into waste water treatment plants. Guanylurea, metformin’s transformation product, is said to be a “dead-end” metabolite that is accumulating in water. However, recent studies found that there is an enzyme, guanylurea hydrolase, in Pseudomonas mendocina strain that is able to break down guanylurea into guanidine and ammonia. To further investigate the use of this enzyme, research was conducted to investigate 13 potential bacteria that have similar enzymes to that of guanylurea hydrolase (GUH). Plasmids containing the genes encoding the guanylurea hydrolase homologs were transformed into E.coli. These cells were induced to stimulate protein production and prepare cell extracts.  Protein concentrations were determined using the BCA assay while enzyme activity was tested by measuring the amount of ammonia released using the Betherlot reaction. The substrate specificity of the guanylurea hydrolase homologs was investigated on guanylurea, as well as similar compounds including, biuret, cyanoguanidine, and 2-imino-4-thiobiuret. Most GUH homologs can only degrade guanylurea, implying that guanylurea hydrolase has a narrow substrate specificity. In summary, these experiments are key to understanding the breakdown of metformin and its transformation product, guanylurea.