While adenoviral (Ad) vectors are the most commonly used gene delivery vector for human gene therapy, improvements must be made to increase Ad specificity to tumors if they are to be used for cancer gene therapy. Our goal is to target adenoviral vectors to breast cancer (BrCa) cells to induce cell killing while reducing toxicity to non-tumor cells. We have generated a non-replicating BrCa-targeted adenoviral vector that utilizes a mammary-specific promoter (MPE2) to drive expression of a therapeutic gene, herpes simplex virus-1 thymidine kinase (HSV-TK). Cells expressing HSV-TK are sensitive to the prodrug ganciclovir (GCV). Thus, AdMPE2TK can induce BrCa-specific cell death when administered in combination with GCV. The expression of HSV-TK also allows cells to be visualized using positron emission tomography (PET) using radiolabeled PET substrates 3’-[18F]fluoro-3’-deoxythymidine ([18F]FLT) and 9-(4-[18F]fluoro-3-hydroxymethylbutyl)-guanine ([18F]FHBG). We first characterized the immunocompetent MTHJ murine breast cancer model as having high biological significance to human breast cancer and reveal its ability to uptake standard PET tracers. We then showed the BrCa specificity of the AdMPE2TK vector to kill BrCa cells in vitro in comparison to the non-specific AdCMVTK vector utilizing the cytomegalovirus promoter. Neither vector was shown to induce in vivo tumor regression, however the AdCMVTK vector caused liver toxicity in immunocompetent mice. In contrast, the AdMPE2TK vector did not induce any measurable toxicity, highlighting its specificity and potential for cancer gene therapy. Finally, using MTHJ murine tumor cells, in vitro cell uptake experiments revealed the ability of AdTK vectors to induce an increase in accumulation of [18F]FHBG. In vivo PET imaging was then used to evaluate the accumulation of [18F]FLT and [18F]FHBG in established MTHJ tumors injected with AdTK vectors, however no increase in accumulation was observed. This thesis outlines the first preclinical evaluation of the BrCa-specific MPE2 promoter to target HSV-TK for cancer gene therapy. We illustrate the ability of AdMPE2TK to induce a therapeutic effect in combination with GCV. In addition, this project represents the first imaging studies of an adenoviral vector utilizing the MPE2 promoter. This research outlines the potential of the MPE2 promoter and illustrates its application in BrCa gene therapy.