Thymidylate kinase (TMPK, EC2.7.4.9) is the enzyme that converts deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) in the synthesis of dTTP, an essential building block of DNA. To date, there is only one gene () known to encode TMPK in mammalian cells. In this study, we investigated the distribution of TMPK activity and protein in subcellular fractions by using activity measurements and by using a specific antibody against -encoded TMPK (canonical TMPK). TMPK activity was detected in all subcellular fractions, of which the mitochondrial outer membrane contained the highest activity. High levels of canonical TMPK protein were detected in the cytosolic fraction, whereas low levels were found in the nuclear and mitochondrial matrix fractions. Strikingly, despite the detection of high TMPK activity in the mitochondrial outer membrane, canonical TMPK protein was not detected in this fraction. These results suggest that the TMPK activity detected in the outer membrane fraction may originate from a novel dTMP kinase, distinct from the canonical TYMK.