Cytidine deaminase (CDA) is the major enzyme of gemcitabine inactivation. The aim of this study was to determine whether the CDA Lys27Gln polymorphism influenced gemcitabine deamination in blood samples from 90 lung cancer patients. The polymorphism was studied with Taqman probes-based assay; CDA activity was evaluated by HPLC in cytoplasmic extracts from red blood cells. Mean enzymatic activity was significantly lower in patients carrying the CDA Lys27Lys than in patients with the Lys27Gln or Gln27Gln protein (P < 0.05). CDA genotyping may be useful in screening patients before gemcitabine treatment, in order to identify subjects with lower CDA activity and potentially better clinical outcomes after gemcitabine-based chemotherapy.