Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host–pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-γ (IFN-γ) and tumor necrosis factor–α (TNF-α) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein–intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-γ. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-α treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-γ–treated organotypic slice cultures was severely impaired compared with untreated and TNF-α–treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-γ and TNF-α exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.