NDP kinase and thymidylate kinase are essential for DNA precursor formation in that they phosphorylate the products of de novo deoxyribonucleotide biosynthesis, deoxyribonucleoside 5′-diphosphates and thymidine 5′-monophosphate to the corresponding triphosphates which then serve as DNA polymerase substrates. The two enzymes have been measured in synchronous cultures of the green algae, S. obliquus. Thymidylate kinase exhibits an activity peak at the 11 -12th hour of the 24-hour cell cycle, coinciding with DNA synthesis. Enzyme activity is markedly stimulated in presence of fluorodeoxyuridine in the culture medium. This behaviour of dTMP kinase is very similar to that of three other S phase-specific peak enzymes previously analyzed in synchronous algae, viz. ribonucleotide reductase, thymidylate synthase, and dihydrofolate reductase. In contrast, NDP kinase exhibits high and constant activity through the entire cell cycle. The two kinases have been isolated from cell-free extracts, and separated from each other by chromatography on Blue Sepharose. The peak enzyme, dTMP kinase, has been purified to near homogeneity and its catalytic properties are described; the molecular weight is 56,000. NDP kinase activity is separable into two enzyme fractions, both of molecular weight 100,000 (or higher), which are unspecific with respect to ribonucleotide and deoxyribonucleotide substrates. Characterization and purification of the whole series of deoxyribonucleotide-synthesizing enzymes from one organism provides a basis for in vitro experiments towards reconstitution of an S phase-specific DNA precursor/DNA replication multienzyme aggregate.