The possible mechanism of eosinophilia was studied in rats undergoing primary infection with Nippostrongylus brasiliensis (Nb). In vivo studies showed that the kinetics of intestinal tissue eosinophilia was not directly related to those of the intestinal worm burden. Furthermore, Nb worm extract has no or only very weak in vitro eosinophil chemotactic activity, suggesting that parasite-derived eosinophil chemotactic factor (ECF) is, if at all, not a major regulator for intestinal tissue eosinophilia in this Nb rat system. On the other hand, when mesenteric lymph node (MLN) cells obtained various days after infection were cultured, potent ECF activity was detected in the cell-free supernatant from the cultures of MLN cells 15-20 days after infection, at which time marked intestinal tissue eosinophilia was observed in vivo. Production of ECF by MLN cells from Nb-infected rats seems to be spontaneous, since these cultures were performed without adding worm antigen. ECF-producing activity of day-20 MLN cells was suppressed by adding various metabolic inhibitors such as cycloheximide, mitomycin C, or puromycin. After Sephadex G-75 gel filtration, ECF activity produced by day-20 MLN cells was associated with two different molecules.