Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.