Deoxycytidine kinase (dCK1) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and anti-viral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/ dephosphorylation, direct demonstration was lacking. Here we show that dCK overexpressed in HEK 293T cells was labelled after incubation of the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labelling of dCK. These results indicate that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labelled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, four in vivo phosphorylation sites were identified: Thr3, Ser11, Ser15 and Ser74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser74 antibody demonstrated that Ser74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser74 was also detected on endogenous dCK in leukemic cells, in which Ser74 phosphorylation state was increased by agents that enhance dCK activity. Our study provides direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights phosphorylation of Ser74 as determining for dCK activity.