We have previously demonstrated that inhibition of expression of amoebapore A (AP-A) by antisense RNA caused a marked decrease in the virulence of the parasite. A four-fold over-expression of AP-A was obtained with plasmid (pA7) which has theap-a gene under the control of geneEhgLE-3-RP-L21. The virulence of the transfected trophozoites, however, was also decreased. Excess of AP-A protein was found in the cytosol and a significant amount was released into the surrounding media. Transfection of the parasite with a plasmid (psAP-1) in which theap-a gene was introduced with its own regulatory sequences, caused a total suppression of the transcription and translation of both the genomic and episomalap-a genes. The silenced transfectant was not virulent at all. These results demonstrate that important factors need to be expressed at the correct cellular location and that the parasite has additional internal control mechanisms such as transcriptional gene silencing which can prevent excess amounts of gene expression.