Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA replication. The only known enzyme performing de novo dTMP synthesis is thymidylate synthase (TS). The goals of this thesis were to provide biochemical evidence for the existence of TS in chlamydiae, to investigate the mechanism by which the parasite obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts dUMP into dTXP. In vitro experiments conclusively establish these findings by the demonstration of TS activity in extracts prepared from host-free chlamydial reticulate bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial strains can synthesize folates de novo; however strains vary significantly in their ability to transport preformed folates from the host cell. A C. trachomatis gene which is capable of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned. Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open reading frame shows absence of sequence homology to known TS. Unique in vitro characteristics shared by the enzyme activities from both chlamydial extract and recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.