200Gy gamma-irradiated Toxoplasma gondii RH tachyzoites failed to reproduce in vitro and in vivo. In short-term cultures, these parasites maintained a respiratory response, the ability to invade cells and preserved protein and nucleic acid synthesis. ELISA and Western blotting techniques demonstrated the similarity in humoral response between mice infected with gamma-irradiated tachyzoites and animals infected with naive parasites and treated with sulfadoxine, higher than mice immunised with formaldehyde-killed tachyzoites. Splenocyte stimulation by T. gondii antigen produced lymphoproliferative response and cytokine profile (IL-10, IL-12, IFN-gamma and TNF-alpha) similar to those produced by chronic natural infection. Mice immunised with irradiated tachyzoites had extended survival times after subsequent tachyzoite challenge, and displayed minimal cerebral pathology after cyst challenge. Irradiated tachyzoites lose their reproductive ability whilst maintaining metabolic function and may provide a novel tool for the study of toxoplasmosis and vaccine development.