The most frequent site of prostate cancer metastasis is the bone. Adhesion to bone-specific factors may facilitate the selective metastasis of prostate cancer to the skeleton. Therefore, we tested whether prostate cancer bone metastasis is mediated by binding to type I collagen, the most abundant bone protein. We observed that only bone metastatic prostate cancer cells bound collagen I, whereas cells that form only visceral metastases failed to bind collagen. To confirm the relationship between collagen adhesion and bone metastatic potential, a collagen-binding variant of human LNCaP prostate cancer cells was derived through serial passage on type I collagen (LNCaP(col)). Fluorescence-activated cell sorting analysis showed that LNCaP(col) cells express increased levels of the integrin collagen I receptor alpha(2)beta(1) compared with LNCaP cells. Antibodies to the alpha(2)beta(1) complex inhibited LNCaP(col) binding to collagen, confirming that integrins mediated the attachment. Correspondingly, LNCaP(col) cells displayed enhanced chemotactic migration toward collagen I compared with LNCaP cells, an activity that could be blocked with alpha(2)beta(1) antibodies. To directly test the role of alpha(2)beta(1)-dependent collagen binding in bone metastasis, LNCaP and LNCaP(col) cells were injected into the tibia of nude mice. After 9 weeks, 7 of 13 (53%) mice injected with LNCaP(col) developed bone tumors, whereas 0 of 8 mice injected with LNCaP cells had evidence of boney lesions. LNCaP(col) cells were found to express increased levels of the metastasis-promoting RhoC GTPase compared with parental LNCaP. We conclude that collagen I attachment mediated by alpha(2)beta(1) initiates motility programs through RhoC and suggest a mechanism for prostate cancer metastasis to the bone.