Transplantation of the human ovarian adenocarcinoma cell line, NIH:OVCAR-3 into athymic mice produces two morphologically distinct tumor cell populations (ascites and solid tumors). In the present study, we isolated both tumor cell phenotypes and investigated their relative malignant potential. Since cytoskeletal and morphological changes correlate with metastatic phenotype, expression of the intermediate-filament protein vimentin was compared between ascites and solid tumors. Ascites tumor cells showed a less differentiated epithelial morphology and concurrently expressed higher levels of vimentin. Ascites cells were more efficient in anchorage independent growth when compared with their solid tumor counterpart. Ascites tumor cells were also highly motile compared with the solid tumor cell population (P = 0.006). Migration of ascites tumor cells was further enhanced by type IV collagen, hyaluronic acid, and chondroitin sulfate A. Solid tumor cells removed from the same animal, however, were not significantly affected by these agents. From these studies, we conclude that ovarian cancer cells present in ascites are phenotypic variants which are highly motile compared with solid tumor cells isolated from the same animal. Ascites tumor cells with increased motility may contribute to peritoneal seeding and metastasis.