In order to clarify the environmental factors modulating cell migration, we investigated the effects of human serum on cell migration, and found that serum from adult donors strongly (by 48%) suppressed the migration of human fetal skin fibroblasts into a denuded area in a cell monolayer. Human serum from old donors inhibited cell migration more strongly than that from adult donors. Next, we investigated the properties of migration-inhibitory activity of human serum and serum proteins in order to identify migration-inhibitory substances. Human serum from adult donors strongly suppressed the migration of human fetal skin fibroblasts, although it stimulated cell proliferation more strongly than fetal bovine serum (FBS), indicating that the inhibitory effects of human serum on cell migration was not due to its toxic effects. The inhibition of cell migration by human serum was concentration dependent. It was demonstrated that the inhibition did not depend on the inhibitory effects of human serum on collagen synthesis. The migration-inhibitory activity was seen in fractions over 100 kDa, as determined by an ultrafiltration membrane, and no inhibitory activity was observed in fractions under 100 kDa. On the other hand, it was not detected either in fractions over 100 kDa or under 100 kDa in FBS. Among the over 100 kDa human serum proteins examined, γ-globulin, α2-macroglobulin, and low density lipoprotein (LDL) suppressed fibroblast migration in a concentration-dependent manner. However, among the three, cell migration-inhibiting activity of γ-globulin almost disappeared when cell migration was conducted in 10% FBS-supplemented medium. These results indicated that α2-macroglobulin and LDL were candidate substances for cell migration-inhibiting activity in human serum.