To determine the genotypes of Toxoplasma gondii strains associated with human toxoplasmosis, we developed a sensitive approach for typing parasites grown from clinical samples by short-term in vitro culture. A newly described nested PCR assay was capable of amplifying genomic DNA from as few as five parasites in the presence of host tissues. Typing was based on DNA polymorphisms at the SAG2 locus, encoding tachyzoite surface antigen p22. Restriction fragment length polymorphisms in PCR-amplified SAG2 products were used to classify strains into one of the three major lineages of T. gondii. This approach was successfully used to determine the genotypes of 68 of 72 samples that had been previously isolated from patients with congenital, cerebral, and disseminated toxoplasmosis. Type II strains of T. gondii were found in a majority of the samples, accounting for 55 (81%) of the 68 toxoplasmosis cases. In contrast, type I and III strains were found in only 7 (10%) and 6 (9%) of the 68 cases, respectively. The results of this study support the previous finding that type II strains are most often associated with human toxoplasmosis. Nested PCR analysis at the SAG2 locus provides rapid assignment of T. gondii to a specific genotype that should be useful in analyzing a variety of clinical samples.