Abstract The establishment of culture conditions suitable for inducing differentiation of Toxoplasma gondii tachyzoites into parasites resembling the latent bradyzoite form has opened this important developmental transition to experimental analysis. In order to develop a genetic marker suitable for positive and negative selection during parasite differentiation, the T. gondii HXGPRT gene was placed under control of 5′ flanking sequences derived from two bradyzoite-specific genes: BAG1 and LDH2. Random transgene integration at undefined genomic loci resulted in modest regulation (≈5–6-fold induction) above relatively high background levels (∼4% of wild-type controls). Integration of transgenes at a defined genomic position was achieved by targeting the uracil phosphoribosyl transferase ( UPRT) locus using flanking homologous sequences and fluorouracil selection. This strategy was found to provide the added advantage of enhancing bradyzoite induction frequencies under conditions of pyrimidine starvation (low CO 2). Constructs integrated in the direction of normal UPRT transcription exhibited moderate levels of inducibility, but transgenes integrated in the opposite direction were dramatically induced under differentiation conditions: 50–100-fold above the very low levels observed in tachyzoites (<1% control). Positive selection (using mycophenolic acid) was shown to inhibit tachyzoites but not bradyzoites, while negative selection (using 8-azahypoxanthine) inhibited bradyzoites only. Stage-specific regulation of the HXGPRT selectable marker should permit genetic selections for the identification of mutants in the bradyzoite differentiation process.