Derivatives obtained by monosubstitution at six positions of thymidine, 5'-amino-5'-deoxythymidine, or 5-bromo-5,6-dihydrothymidine have been studied as inhibitors of Escherichia coli and hamster thymidine kinases (TK). Affinity for the enzymatic thymidine binding sites was assessed from apparent enzyme-inhibitor dissociation constants (K, values; for inhibitions competitive with respect to thymidine at near-saturating ATP levels) or Is0 values (for noncompetitive inhibitions). To provide indices of relative affinity for each enzyme, the K, and 1% values were divided by the appropriate KM value (33 or 3.3 FM) of thymidine with the E. coli and hamster enzymes, respectively. 3-Amylthymidine gave I50/KM = 20 with E. coli and K,/KM = 21 with hamster TK; 5-amino-2'-deoxyuridine gave I50/KM = 840 with E. coli TK and KJKM = 135 with hamster TK; tr~ns-5-bromo-6-ethoxy-5,6-dihydrothymidine diastereoisomers at 16 mM showed almost no inhibition of E. coli TK and gave K, = 0.2-0.3 mM with hamster TK; 3'-acetamido- and 3'-(ethylthio)-3'-deoxythymidines gave IbO/KM = 183 and 9.6, respectively, with E. coli TK and K,/KM = 750 and 3.6, respectively, with hamster TK; 5'-C-(acetamidomethyl)- and 5'-C-(propionamidomethy1)thymidine epimers inhibited both enzymes competitively (KJKM = 26-198 for E. coli and 20-330 for hamster), and the extra methyl present in the propionamido derivatives produced 7.5- and 9-fold differential effects on binding; 5'-amino-5'-deoxythymidine also inhibited competitively (K,/KM = 9.6 for E. coli and 1.8 for hamster), and addition of a 5'-N-hexyl group reduced the differential affinity (KI/KM = 78 for E. coli and 54 for hamster); some 5'-(alkylthio)-5'-deoxythymidines inhibited hamster TK competitively but activated E. coli TK, possibly by interacting at its dCDP-dCTP activator site. The evidence indicates that thymidine derivatives suitably substituted at any one of the above six positions can bind to the thymidine sites of the E. coli and hamster thymidine kinases in a species-selective manner.