Nonspecific toxicity and resistance to traditional cytotoxic drugs are impediments to effective cancer therapy. Development of drugs targeting cellular molecules that mediate malignant characteristics may improve therapy. Antisense drugs that reduce mRNA and protein on which tumor cells depend for viability and treatment resistance are examples of such candidates. In particular, combining antisense drugs to simultaneously reduce multiple mRNAs/proteins is predicted to enhance antitumor effects. We hypothesized that combined treatment with silencer RNAs (siRNAs) targeting molecules mediating both proliferation (thymidylate synthase; TS) and survival (Bcl-2) would decrease proliferation and sensitize human tumor cells to nonantisense drugs in a greater-than-additive manner. We report that simultaneous treatment of human cervical carcinoma (HeLa) and breast tumor (MCF-7) cell lines with siRNAs targeting both TS and Bcl-2 had unexpected, nonreciprocal antagonistic effects. Two siRNAs targeting different Bcl-2 mRNA sequences reduced the capacity of TS siRNA to reduce TS mRNA and protein, with no evidence of converse effects by TS siRNA on Bcl-2 mRNA or protein. Moreover, treatment of HeLa cells with siRNA targeting Bcl-2 resulted in increased TS mRNA and protein. Pretreatment of HeLa and MCF-7 cells with TS siRNA sensitized cells to TS-targeting drugs, but addition of antagonistic Bcl-2 siRNA to the pretreatment regimen abrogated sensitization. Combined targeting of separate physiological pathways by antisense reagents may be a useful approach in treatment of cancer, but antagonistic interactions could abrogate advantages or reduce effectiveness of other antisense and nonantisense reagents.