We tested the virulence in mice of Toxoplasma gondii RH strain tachyzoites containing various copies of the chloramphenicol acetyl transferase-herpes simplex virus thymidine kinase fusion sequence (CAT-HSTK). Tachyzoite isolates containing five copies of the fusion sequence were not lethal to female CD-1 outbred or BALB/c inbred mice, at doses up to 10⁶ parasites, while the parental RH strain caused 100% mortality within 2 weeks at doses as low as 10 parasites. Mice infected with CTK11, an isolate containing five copies of the fusion sequence, showed no overt symptoms of disease and were protected from lethal challenge with the parental RH strain. The CTK11 isolate showed no difference in growth rate, the rate of host cell invasion, or extracellular viability in cell culture compared with parental RH parasites, demonstrating that the CAT-HSTK fusion protein does not affect the normal viability of this isolate. B11, B11C, and D1 isolates contained one or two copies of the CAT-HSTK coding sequence, were not sensitive to thymidine in cell culture, and caused 100% mortality in CD-1 outbred mice in <12 days. A fourth isolate, D1C, contained seven copies of the CAT-HSTK fusion sequence and was sensitive to exogenous thymidine (50% inhibitory concentration = 5.5 µM). Mice infected with D1C showed no symptoms of disease and survived beyond 90 days, thus correlating increased CAT-HSTK gene copies with thymidine sensitivity in cell culture and attenuated virulence in mice. BALB/c mice containing a targeted disruption of the gamma interferon gene (gko) were also susceptible to infection with CTK11 parasites but could be rescued by administration of subcutaneous thymidine once each day for 5 or 10 days following infection. These results suggest that the attenuation of CAT-HSTK⁺ isolates in mice is directly due to active thymidine kinase that likely alters the pyrimidine biosynthetic pathway in these parasites.