We established an orthotopic treatment model of prostate cancer to generate reproducible primary and metastatic carcinoma in immunocompetent C57BL/6 mice. Using an in vivo selection scheme of intraprostatic implantation of TRAMP‐C1 cells, primary prostate tumors were cultured and recycled three times by intraprostatic injection resulting in the selection and establishment of the recycled cell line TRAMP‐C1P3. Prostate tumors were detected ∼30 days post‐implantation with periaortic lymph node metastasis in 19/20 (95%) of mice. Tissue culture amplification, DNA ploidy and PCR amplification of the SV40 transgene were used to detect metastatic TRAMP‐C1P3 in lymph node specimens. Tissue culture amplification and DNA ploidy were as sensitive as SV40 transgene amplification by PCR in detection of early metastatic disease in draining lymph nodes. To establish the use of the orthotopic model of prostate cancer for immunotherapy, mice were injected orthotopically with TRAMP‐C1P3 cells and 7 days post‐implantation treated daily for 28 days with either flt3L or carrier control. Carrier‐treated mice had clinically detectable prostate tumors, lymph node metastasis and were moribund at 29–35 days, whereas flt3L therapy markedly suppressed primary TRAMP‐C1P3 growth and lymph node metastasis, and prolonged survival. In summary, we have established a reproducible and clinically relevant orthotopic treatment model of prostate cancer in immunocompetent mice with application to a variety of therapeutic strategies. We demonstrate that flt3L treatment suppressed orthotopic prostate tumor growth and lymph node metastasis reinforcing a role for flt3L as an immunotherapeutic strategy for prostate cancer. © 2003 Wiley‐Liss, Inc.