During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd ؊ ) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd ؊ phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.