5-Fluorouracil (5-FU) has been one of the most commonly used drugs in the treatment of various types of cancer (in particular, colorectal and breast) for the past fifty years. 5-FU is a drug for which genetic and epigenetic variation in the drug-metabolizing enzyme (dihydropyrimidine dehydrogenase [DPD]) and the drug target enzyme (thymidyalate synthase [TS]) can influence both toxicity and response to treatment. Through a comprehensive investigation and screening of various genetic (SNPs, deletions, insertions and duplications) and epigenetic (methylation in the promoter region) variations in the six major genes (DPYD, DHP, BUP, TP, OPRT and TS) encoding 5-FU metabolizing enzymes, we intend to identify the spectrum of molecular alterations associated with a patient’s response to the drug. We will then measure the frequency of known and newly observed mutations in patients with adverse side effects, healthy volunteers and various Israeli populations. The following step will be to establish a set of mutations that is likely to more fully elucidate the causes that impact the toxicity and efficacy of 5-FU. Finally, we will translate our research findings into a genetic testing tool for use prior to cancer treatment, thus integrating Pharmacogenetics into health care practice.