We have previously shown that the toxic pro-oxidant methylselenol is released from selenomethionine (SeMET) by cancer cells transformed with the adenoviral methionine alpha,gamma-lyase (methioninase, MET) gene cloned from Pseudomonas putida. Methylselenol damaged the mitochondria via oxidative stress, and caused cytochrome c release into the cytosol thereby activating caspase enzymes and thereby apoptosis. However, gene therapy strategies are less effective if tumor cells overexpress the antiapoptotic mitochondrial protein bcl-2. In this study, we investigated whether rAdMET/SeMET was effective against bcl-2-overproducing A549 lung cancer cells. We established two clones of the human lung cancer A549 cell line that show moderate and high expression levels of bcl-2, respectively, compared to the parent cell line, which has very low bcl-2 expression. Staurosporine-induced apoptosis was inhibited in the bcl-2-overproducing clones as well as in the parental cell line. In contrast to staurosporine, apoptosis was induced in the bcl-2-overproducing clones as well as the parental cell line by AdMET/SeMET. Apoptosis in the rAdMET-SeMET-treated cells was determined by fragmentation of nuclei, and release of cytochrome c from mitochondria to the cytosol. A strong bystander effect of AdMET/SeMET was observed on A549 cells as well as the bcl-2-overproducing clones. rAdMET/SeMET prodrug gene therapy is therefore a promising novel strategy effective against bcl-2 overexpression, which has blocked other gene therapy strategies.