Besides a fully processed, secreted form of clusterin (sCLU), an alternative proapoptotic form of the protein targeting the nucleus (nCLU) was recently described. The possible differential roles played by the two clusterin forms in growth and motility of nonmalignant and malignant prostate cells are investigated here. sCLU or nCLU was transiently transfected in both androgen-independent prostate cancer cells (PC3 and DU 145) and immortalized prostate epithelial cells (PNT1A, a nontumoral control). Then, cell growth, motility, and cytoskeleton organization were studied. We found that (a) in PNT1A cells, both sCLU and nCLU significantly decreased cell proliferation and motility; (b) in PC3 and DU 145 cancer cells, only nCLU inhibited cell growth and migration, with sCLU being ineffective; and (c) the antimotility effect of nCLU was accompanied by a dramatic dismantling of the actin cytoskeleton. Moreover, transfection with "full-length" CLU cDNA produced both sCLU and nCLU in nonmalignant PNT1A cells, whereas only sCLU was found in cancer cells. Thus, CLU gene expression might play a crucial role in prostate tumorigenesis by exerting differential biological effects on normal versus tumor cells through differential processing of CLU isoforms in the two cell systems. We also found that nCLU binds to alpha-actinin, a key protein for the regulation of actin cytoskeleton, and that nCLU and alpha-actinin colocalize in the cytoplasm. Thus, the antimotility activity of nCLU and its ability to cause dismantling of the actin cytoskeleton seem to be mediated by its binding to alpha-actinin.