Insights into the molecular basis of hormone-refractory prostate cancer have principally relied on human prostate cancer cell lines, all of which were derived from patients who had already failed hormonal therapy. Recent progress in developing genetically engineered mouse prostate cancer models provides an opportunity to isolate novel cell lines from animals never exposed to hormone ablation, avoiding any potential bias conferred by the selective pressure of the castrate environment. Here we report the isolation of such a cell line (Myc-CaP) from a c-myc transgenic mouse with prostate cancer. Myc-CaP cells have an amplified androgen receptor gene despite no prior exposure to androgen withdrawal and they retain androgen-dependent transgene expression as well as androgen-dependent growth in soft agar and in mice. Reexpression of c-Myc from a hormone-independent promoter rescues growth in androgen-depleted agar but not in castrated mice, showing a clear distinction between the molecular requirements for hormone-refractory growth in vitro versus in vivo. Myc-CaP cells represent a unique reagent for dissecting discreet steps in hormone-refractory prostate cancer progression and show the general utility of using genetically engineered mouse models for establishing new prostate cancer cell lines.