The synthesis of deoxythymidine triphosphate (dTTP), as one of the four building blocks ofDNA, plays a crucial role for the plant. Each cell compartment with its own sub genometherefore has a subset of enzymes necessary for the formation of thymidylates. However, therespective compartments are not purely self-sufficient systems, but are in constant exchangeand can support each other depending on the developmental stage. Furthermore, thecontribution of individual steps of thymidylate metabolism to the thymidylate pools varies inimportance over time.According to current knowledge, the essential enzymes involved in the synthesis of dTTP inArabidopsis thaliana are Dihydrofolate Reductase-Thymidilate Synthase (DHFR-TS),Thymidine Kinase (TK) and Deoxythymidine Monophosphate Kinase (TMPK, ZEU1). Completeloss of these enzymes is lethal for the plant, or in the case of TK, leads to a plant that can nolonger reproduce. However, in the case of DHFR-TS and TK, only one of the two homologuesis required for the development of a viable plant.This work shows that at germination, de novo biosynthesis, which proceeds via DHFR-TS, playsa minor role compared to salvage. However, the exclusively mitochondrial DHFR-TS2isoenzyme appears to be more important than its cytosolic counterpart DHFR-TS1. A similarpicture emerges for salvage. TK1b, present in chloroplasts and mitochondria, plays a moreimportant role than its cytosolic representative TK1a. Particularly the chloroplastic TK1b has agreater impact in supplying the cell with thymidylates, which coincides with the synthesis ofcpDNA being also the greatest sink during this time. A TK1b mutant shows a severe arrest incpDNA synthesis during germination. The synthesis of cpDNA is partially restored in theestablished seedling and thymidylate pools are then also affected in a mutant lacking cytosolicTK1a suggesting that the supply of thymidylates is then secured by both cytosolic andorganellar salvage.Among the less important enzymes, appear to be dUTP Pyrophosphatase 1 localized inchloroplasts and mitochondria (DUT1org) and dCMP Deaminase (DCD) localized inmitochondria. Both enzymes play a minor role in providing dUMP for DHFR-TS. In neither ofthe two associated mutants a serious disruption of thymidylate formation is observable. Thismay be due to an involvement of both enzymes in the formation of dUMP, as indicated bypreliminary data suggesting that a DCD DUT1org mutant is not viable.