Cats are important in the epidemiology of Toxoplasma gondii because felids are the only definitive hosts that can excrete environmentally resistant oocysts. Fresh samples of brain from 103 Spanish cats with antibodies to T. gondii were analyzed for T. gondii DNA using nested-PCR; 47 (45.5%) were found to be positive. Further characterization of DNA from 46 cats using RFLP-PCR at the 3′ and 5′ ends of the SAG2 locus revealed that 12 (26%) isolates were Type I and 34 (74%) were Type II; no Type III were found, and the 47th sample could not be classified to its genetic type. In addition, T. gondii was also isolated by bioassay in mice from 42 of 103 seropositive cats. This is the first report of T. gondii characterization from cats in Spain.