Following SV40 infection of CV-1 monkey cells, cytosol thymidine (dT) kinase activity increased 4- to 10-fold. This enzyme was not significantly different from the cytosol dT kinase of uninfected cells with respect to mobility, isoelectric point, sedimentation coefficient, and phosphate donor specificity. Monkey mitochondria contain a distinctive dT kinase isozyme localized in the mitochondrial matrix. The distinctive mitochondrial isozyme had a higher electrophoretic mobility, lower isoelectric point, and smaller sedimentation coefficient than the cytosol dT kinase, and could utilize either ATP or UTP as phosphate donor; it did not increase after SV40 infection. Mitochondria also contained a cytosol-like dT kinase which was enhanced in infected cells. These findings suggest that SV40 infection derepresses the normal cytosol dT kinase of host cells.