Deoxycytidine (CdR) kinase (E.C. 2.7.1.74) acts as a salvage enzyme in DNA biosynthesis, but little is known about this important nucleoside kinase in the brain. We report that CdR kinase activity in the 100,000 x g cytosolic fraction of normal adult rat brain cortex was 0.89 +/- 0.04 nmol/hr/mg protein which is twice that of the liver enzyme. For brain CdR kinase the apparent Km for CdR, ATP and Mg++ were 0.22, 1.1 and 0.63 mM, respectively. When the cytosolic preparation was incubated at 37 degrees C, CdR kinase activity rapidly decreased (t1/2 = 15 min); CdR (400 microM) protected the enzyme. Addition of DFDC (0 to 100 microM) competitively inhibited brain CdR kinase activity with Ki = 17 microM. DFDC elevated the apparent Km for CdR of brain CdR kinase 3.5-fold, from 0.22 to 0.8 mM. DFDC did not inhibit brain TdR kinase. AZT, which competitively inhibited TdR kinase (Ki = 0.6 microM), did not affect brain CdR kinase activity. These results indicate that the cytosol of rat brain contains CdR kinase which is inhibited by the deoxycytidine analog, DFDC. The enzyme is protected from thermal denaturation by CdR but not by TdR.