In the protozoan parasite Toxoplasma gondii, cell division occurs by an unusual internal budding process whereby two daughter cells develop within and eventually subsume the mother cell. We have examined this process using inhibitors targeted at specific events in the cell cycle. By adding inhibitors to newly established parasites we were able to examine the effects of the inhibitors on parasites treated at the start of intracellular development and many hours prior to the onset of daughter cell budding. As with other eukaryotes, inhibitors of nuclear DNA synthesis blocked parasite DNA synthesis and prevented cell division. Examination of parasites treated with the nuclear DNA synthesis inhibitor aphidicolin showed that the formation of daughter apical complexes and the initiation of budding occurred as normal and only the inability of the nucleus to become incorporated into the daughter cells prevented successful cell division. Moreover, these inhibitory effects of aphidicolin were not reversible. The initiation of nuclear DNA synthesis and cell division in newly invaded Toxoplasma required both gene transcription and protein synthesis, although inhibitors of mitochondrial DNA synthesis, transcription and protein synthesis did not block parasite division. Thus, unlike most eukaryotes, Toxoplasma tachyzoites have separated nuclear DNA replication and mitosis from the events associated with cell division (daughter cell budding). This implies that Toxoplasma tachyzoites may have dispensed with specific cell cycle checkpoints present in other eukaryotes with, in particular, a DNA-replication checkpoint control either missing, or downregulated in this stage of the parasite life cycle.