Investigations on deoxythymidine Kinase show that: (a) 5-ethyl-2′-deoxyuridine-β (EDU-β) binds to about 100-fold weaker (Km 2.7 × 10-4) than deoxythymidine (Km 2.5 × 10-6) and the rate of its phosphoration (0.7 nmol/mg protein/15 min) is about the half of the deoxythymidine (1.52 nmol/mg protein/15 min); (b) 5-ethyl-2′-deoxyuridine-α (EDU-α), 5-propyl-2′-deoxyuridine (α and β) as well as α and β-5-isopropyl-2′- deoxyuridines do not serve as substrates for this enzyme.