Lymphokine-containing supernatants have the ability to inhibit the migration of a variety of tumor cells in vitro in the absence of cytotoxic effects. In the present study, this tumor migration inhibition activity has been characterized in order to determine whether the responsible factor is the same or different from other known migration-inhibitory lymphokines. Since no preparations purified to homogeneity are generally available for these various factors, a variety of indirect procedures is necessary to make this determination. The profile of effects of monosaccharides, protease inhibitors, and neuraminidase on tumor migration inhibition factor, taken in conjunction with previously reported studies of other physicochemical and biological properties, provides evidence that tumor migration inhibition factor is distinct from both migration inhibition and leukocyte-inhibitory factor. A lymphokine with migration-inhibitory activity against tumor cells is a good candidate for a variety of protective functions in vivo.