We have analyzed the mechanisms controlling the accumulation of cytotoxic/suppressor T lymphocytes in tumor tissues. We found that tumor-infiltrating helper/inducer T cells isolated from T-9 gliosarcoma-sensitized rats between 4 and 6 days after T-9 gliosarcoma inoculation produced a lymphocyte migration factor (LMF) during in vitro culture. Four peaks of LMF activity (A through D) were detected upon fractionation of LMF by using a Mono Q anion exchange column chromatography. Peak C exhibited the strongest activity among the four peaks of LMF. The action of peak C was chemotactic, but not chemokinetic. Peak C had an isoelectric point of 8.0 and a Mr of 26,000 Da. Only cytotoxic/suppressor T cells were found to be sensitive to peak C in vitro as well as in vivo. It is thus likely that peak C is responsible for the infiltration of cytotoxic/suppressor T cells into tumor tissues. The infiltration of lymphocytes into tumor tissues might also be regulated by the expression of lymphocyte sensitivity for LMF. The target molecule for LMF at 4 days may involve an asparagine-linked oligosaccharide.