Dihydropyrimidine dehydrogenase (DPD) degrades over 80% of administered 5-fluorouracil (5FU), thereby regulating the efficacy of this commonly used anticancer agent. DPD activity is highly variable (8-21-fold) and individuals with reduced activity have a high risk of 5FU toxicity. DPYD encodes DPD protein and 13 different mutations have been reported in DPD-deficient subjects. However, the contribution of these variant genotypes to polymorphic DPD activity in vivo is not clear. The previously described DPYD mutations are contained in 10 exons. These 10 exons were sequenced in a cohort of cancer patients with reduced (n = 23) or normal (n = 14) DPD activity to determine the contribution of each variant allele to low DPD activity in vivo. Eight of the 13 previously defined DPYD mutations (G62A, delta TCAT295-298, C703T, G1003T, G1156T, delta C1897, G2657A, and G2983T) were not detected. A previously defined exon 13 mutation (G1601A) was detected in three individuals with reduced DPD activity. An exon 14 splice donor site mutation (intron14 G1A) was detected in a normal DPD activity individual. It was demonstrated that T85C, A1627G and G2194A are common polymorphisms. A novel exonic mutation (T1679G) was detected in a patient with reduced DPD activity and 5FU toxicity. In addition, three novel common polymorphisms were detected in introns 10 and 13. Only three patients did not have any mutations and 30 had multiple DPYD mutations in the regions examined. However, only 17% (4/23) of the patients with a low DPD phenotype have a molecular basis for reduced activity. Although novel DPYD variants have been identified in this study, the 17 DPYD mutations now described do not entirely explain polymorphic DPD activity and toxic response to 5FU. These data emphasize the complex nature of the molecular mechanisms controlling polymorphic DPD activity in vivo.