We investigated whether mediators released from rat splenic mononuclear cells could control the in vitro migration of nonsensitized resting rat lymphocytes. Rat splenocytes stimulated with concanavalin A, other mitogens, or histamine release three lymphokines that alter rat lymphocyte migration. A positive chemokinetic factor, termed lymphocyte chemoattractant factor (LCF), has a molecular weight (MW) between 50 and 70 kDa. Two negative chemokinetic lymphokines can also be identified; lymphocyte migration inhibitory factor (LyMIF, MW 25-45 kDa) and a high MW inhibitor (HWMI, MW greater than 70 kDa). Lymphokines were destroyed by heat as well as by treatment with neuraminidase and trypsin. The action of LCF and LyMIF was prevented by phenylmethylsulfonylfluoride, a specific serine esterase inhibitor, and the action of LyMIF was also blocked by alpha-L-fucose. The discovery of these mediators provides the opportunity to study the importance of such chemokinetic lymphokines in animal models of disease.