LNCaP lineage‐derived human prostate cancer cell lines C4‐2 and C4‐2B₄ acquire androgen independence and osseous metastatic potential in vivo. Using C4‐2 and C4‐2B₄ the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate‐specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4‐2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4‐2 in athymic hosts produced spinal metastases in 1/5 animals at 8–12 weeks post‐injection; PC‐3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4‐2 failed to establish tumor colonies. Intrailiac injection of C4‐2 but not LNCaP nor C4‐2B₄ cells in athymic mice established rapidly growing tumors in 4/8 animals at 2–7 weeks after inoculation. Intrafemoral injection of C4‐2 (9/16) and C4‐2B₄ (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3–12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4‐2 (8/8) and C4‐2B₄ (8/8) cells formed PSA‐producing, osteoblastic tumors in the bone marrow space within 3–5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription‐polymerase chain reaction (RT‐PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA‐producing and osteoblastic human prostate cancer xenograft model in mice. Int. J. Cancer 77:887–894, 1998.© 1998 Wiley‐Liss, Inc.