The virulence of the oocysts of 7 Czech Toxoplasma gondii isolates was tested. The oocysts were obtained by experimental infection of cats with the tissue cysts of T. gondii isolates from dogs, cats, and rabbits. The cats shed the oocysts in feces, with prepatent periods of 3–5 days postinfection (PI); the patent period was 7–18 days. The number of oocysts shed varied between 0.94 million and 47 million, with 0.66 million–39 million oocysts found in the daily samples of excrement. The cats ceased oocyst production at 11–22 days PI. Sporulated oocysts were used to prepare infective doses of 1 to 10⁵ oocysts for oral infection of 10 mice. Deoxyribonucleic acid isolated from 4 T. gondii isolates was used in polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) for amplification of the ROP1 gene and restriction of the product of amplification by restriction endonuclease DdeI. On the basis of their biological characteristics, all 7 isolates belonged to the group of “avirulent” strains. In the PCR-RFLP tests, 2 isolates, K9 and K19, showed an “avirulent” strain pattern.