Derivatives obtained by single replacements or substitutions of groups at eight positions of thymidine (TdR) have been examined as inhibitors of rat mitochondrial (M-TK) and cytoplasmic (C-TK) isozymes of thymidine kinase. A C-TK (pI = 7.5) and an M-TK (pI = 5.1) from rat spleen were purified to apparent isozymic homogeneity by isoelectric focusing. Affinities relative to that of TdR for the TdR sites of the isozymes were derived by dividing the Michaelis constants of TdR by the inhibition constants. Of the eight types of TdR derivatives, five had higher affinity for the M-TK site and two had higher affinity for the C-TK site. The most potent and/or selective inhibitors were 3'-O-benzyl-TdR (affinity for M-TK relative to TdR, 100%; differential affinity for M-TK vs. C-TK, 7.5), 5-amino-2'-deoxyuridine (relative affinity for M-TK, 11%; differential affinity affinity for M-TK, 26), 5'-amino-5'-deoxy-TdR (relative affinity for C-TK, 67%; differential affinity for C-TK, greater than 25). Effects of modifying certain of the substituents indicate that at least some of these TdR derivatives are potential progenitors of TK inhibitors of higher potency and selectivity.