ABSTRACT During Toxoplasma gondii infection, a fraction of the multiplying parasites, the tachyzoites, converts into bradyzoites, a dormant stage, which form tissue cysts localized mainly in brain, heart, and skeletal muscles that persist for several years after infection. At this stage the parasite is protected from the immune system, and it is believed to be inaccessible to drugs. While the long persistence of tissue cysts does not represent a medical problem for healthy individuals, this condition represents a major risk for patients with a compromised immune system, who can develop recrudescent life-threatening T. gondii infections. We have investigated for the first time the dynamics and the kinetics of tachyzoite-to-bradyzoite interconversion and cyst formation in vivo by using stage-specific bioluminescent parasites in a mouse model. Our findings provide a new framework for understanding the process of bradyzoite differentiation in vivo. We have also demonstrated that complex molecules such as d -luciferin have access to tissue cysts and are metabolically processed, thus providing a rationale for developing drugs that attack the parasite at this developmental stage.