We used the bioluminescent human prostate carcinoma cell line PC-3M-luc-C6 to non-invasively monitor in vivo growth and response of tumors and metastasis before, during and after treatments. Our goal was to determine the utility of a luciferase-based prostate cancer animal model to specifically assess tumor and metastatic recurrence in vivo following chemotherapy. Bioluminescent PC-3M-luc-C6 cells, constitutively expressing luciferase, were implanted into the prostate or under the skin of mice for primary tumor assessment. Cells were also injected into the left ventricle of the heart as an experimental metastasis model. Weekly serial in vivo images were taken of anesthetized mice that were untreated or treated with 5-fluorouracil or mitomycin C. Ex vivo imaging and/or histology was used to confirm and localize metastatic lesions in various tissues initially detected by images in vivo. Our in vivo data detected and quantified early inhibition of subcutaneous and orthotopic prostate tumors in mice as well as significant tumor regrowth post-treatment. Local and distal metastasis was observed within seven days following intracardiac injection of PC-3M-luc-C6 cells. Differential drug responses and metastatic tumor relapse patterns were distinguished over time by in vivo imaging depending on the metastatic site. The longitudinal evaluation of bioluminescent tumor and metastatic development within the same cohorts of animals permitted sensitive and quantitative assessment of both primary and metastatic prostate tumor response and recurrence in vivo.