Engineered delta ribozymes or HDV ribozymes were used as gene expression modulators in Toxoplasma gondii. The substrate recognition sequence of the trans-acting delta ribozyme, which was derived from the self-cleaving motif located on the antigenomic strand of the hepatitis delta virus genome, was modified to target T. gondii transcripts. The mRNA encoding two well-documented genes, namely uracil phosphoribosyltransferase (UPRT) and hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT) of T. gondii were chosen as the targets for the ribozymes. UPRT and HXGPRT are the operative enzymes of the pyrimidine and purine salvage pathway, respectively. The knockdown of UPRT or HXGPRT expression by the engineered ribozymes resulted in parasites with lower levels of the corresponding transcripts and diminished their abilities to assimilate radioactive pyrimidine or purine analogs. Five out of six engineered ribozymes could cleave their substrates and gave rise to the products, which were detected by primer extension assays. Upon electroporation of individual active ribozymes against the UPRT gene, the uracil incorporation was decreased. Similarly, the ribozymes against HXGPRT caused decreased incorporation of hypoxanthine. When the most active ribozyme against UPRT was used in a combination with the best HXGPRT specific ribozyme, the incorporation of both uracil and hypoxanthine were decreased. Northern blot analysis revealed that the target transcripts were lowered to an undetectable level when specific ribozymes were used, and that the transcripts of the housekeeping gene remained intact. The ribozyme system should thus prove to be effective for the study of gene expression in T. gondii.