We have constructed a genomic DNA library of the Toxoplasma gondii ZS2 strain and isolated a specific cloned DNA sequence from this organism. The restriction map of this cloned 1.1-kb DNA fragment was analyzed. Southern and dot-blot analyses showed that the 32P-labeled DNA fragment hybridized to parasite DNA, to DNAs from peripheral blood leukocytes and the thymus of baby pigs that were artificially infected with T. gondii, and to DNAs of T. gondii-positive anencephalic and hydrocephalic fetuses. It did not hybridize with DNA from controls, (i.e., normal human and baby pig peripheral blood leukocytes, spleen of normal mice, Plasmodium falciparum, Pneumocystis carinii, and pBR322). As few as 100 T. gondii parasites or 500 pg of purified DNA from T. gondii can be detected by dot-blot hybridization. This probe method was specific and sensitive, and has been used successfully in detecting various clinical cases of toxoplasmosis with T. gondii.