5-(Ethylamino)- and 5-acetamido-2'-deoxyuridine 5'-triphosphates were synthesized; the extent and concentration dependence of their inhibitory action on the title enzyme resembled that of the feedback inhibitor TTP. This and other findings provide a tentative indication that bulk tolerance near C-5 of the thymine ring may be more extensive at the TTP site than at the thymidine site. Enzyme-inhibitor dissociation constants (Ki values) were determined for thymidine derivatives monosubstituted at various positions. Competitive inhibition with respect to thymidine (indicative of substituent tolerance in the enzyme-thymidine complex) was produced by 3-amylthymidine (Ki = 65 muM), trans-5-bromo-6-ethoxy-5,6-dihydrothymidine diastereoisomers (Ki = 180 and 310 muM), 5'-C-(acetamidomethyl)- and 5-C-(propionamidomethyl)thymidine epimers (Ki range 65--1100 muM), 3'-acetamido- and 3'-(ethylthio)-3'-deoxythymidines (Ki = 2.5 mM and 12 muM, respectively), and certain 5'-(alkylamino)- and 5'-(alkylthio)-5'-deoxythymidines (Ki range 180--1200 muM). Evidence indicates that bulk tolerance at some, if not most, of the above atoms of thymidine is found in the enzyme-thymidine complexes of human and other mammalian thymidine kinases; attachment of suitable substituents to such atoms could, in principle, lead to thymidine site directed isozyme-specific inhibitors of human cytoplasmic thymidine kinase, which is a candidate target in the design of antineoplastic drugs.