Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates. The purpose of the present study was to determine whether Chlamydia trachomatis obtains deoxyribonucleotides from the host cell. The study was aided by the finding that host and parasite DNA synthesis activity could be distinguished by their differing sensitivities to aphidicolin and norfloxacin. Results from isotope incorporation experiments indicated that any nucleobase or ribonucleoside that could serve as a precursor for host DNA synthesis could also be utilized by C. trachomatis for DNA replication. C. trachomatis utilized only those precursors which the host cell converted to the nucleotide level. Pyrimidine deoxyribonucleotides were efficient precursors for host DNA synthesis; however, they were not used by C. trachomatis. On the other hand, purine deoxyribonucleosides are rapidly catabolized by host cells, it is necessary to regulate their metabolism to determine whether they serve as direct precursors for C. trachomatis DNA synthesis. This was partially achieved by using a hypoxanthine-guanine phosphoribosyltransferase-negative cell line and using deoxycoformycin and 8-aminoguanosine as inhibitors of (deoxy)adenosine deaminase and purine nucleoside phosphorylase, respectively. The results indicated that purine deoxyribonucleosides are efficiently utilized for host cell DNA synthesis even if degradation pathways are inhibited and salvage to ribonucleotides is minimized. In sharp contrast, the purine deoxyribonucleosides were utilized by C. trachomatis as precursors for DNA synthesis only when host catabolic pathways and salvage reactions were intact. High-pressure liquid chromatographic analysis of nucleotide pools extracted from host cells pulsed with radiolabeled precursors suggests that infected cells transport and phosphorylate all deoxynucleosides as effectively as mock-infected control cultures. In aggregate, these results show that chlamydiae do not take up deoxyribonucleotides from the host cells.