Cell motility has been associated with metastatic ability in the Dunning R3327 rat prostatic adenocarcinoma model. Cancer cell motility promoters but not inhibitors have been described by many investigators. Serum-containing and serum-free media conditioned by the nonmotile, nonmetastatic G Dunning subline inhibited the motility of the highly motile, highly metastatic MAT-LyLu subline. Motility inhibition by the G subline-conditioned serum-free media was lost upon heating to 100 degrees C and by treatment with trypsin. The motility inhibitory protein(s) had a molecular weight exceeding 50,000 as determined by diafiltration. G subline-conditioned RPMI 1640 contained several proteins with molecular weights of between approximately 53,000 and 116,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these bands may represent the first inhibitor of cancer cell motility identified.