Abundance of calcitonin (CT) and calcitonin receptor (CTR) mRNA in primary prostate tumors positively correlates with tumor grade, and exogenously added CT increases the invasion of prostate cancer cell lines. We examined acute and chronic actions of CT on migration of highly metastatic PC-3M cells and poorly invasive LNCaP cells on several extracellular matrices in a spheroid disaggregation/migration assay. While PC-3M spheroids displayed maximum disaggregation/migration on vitronectin (VN), LNCaP spheroids preferred collagen but also migrated significantly on VN. Up-regulation of CT significantly enhanced disaggregation/migration of PC-3M spheroids on VN, but not on fibronectin. In contrast, down-regulation of CT, CTR, protein kinase A or urokinase-type plasminogen activator receptor (uPAR) led to amelioration of PC-3M spheroid disaggregation/migration. CT selectively increased surface activity of alpha v beta 3 or alpha 6 beta 5 integrins in PC-3M and LNCaP cell lines, respectively, and uPAR-integrin association. Finally, either CT or urokinase could completely restore migration of CT-knock-down PC-3M spheroids. But, only forced expression of urokinase receptor coupled with exogenous addition of urokinase restored migration of CTR-knock-down spheroids. These results support our hypothesis that up-regulation of CT biosynthesis and activation of CT-CTR axis in primary prostate tumors may have direct relevance in their progression to the metastatic phenotype.