"Transfer factor" was prepared by Sephadex G-25 chromatography of lymph node cell lysates from guinea pigs immunized with ovalbumin or bovine gamma globulin. Treatment of nonimmune peritoneal exudate cells with the transfer factor and specific antigen leads to inhibition of miration of the cells, whereas cells treated with the transfer factor alone or specific antigen alone are not inhibited from migrating. An average of 24-28% inhibition is observed in the presence of transfer factor and specific antigen, but only 5-15% inhibition in the presence of transfer factor and nonspecific antigen. The guinea pig transfer factor we have tested in vitro has some physical characteristics in common with human transfer factor.Transfer factor is defined as the dialyzable portion of human leukocyte extracts that transfers cellular immunity from immune to nonimmune individuals. This material is prepared from white blood cells of donors with positive skin tests and tested by injection into recipients with negative skin tests. At some later time, the recipients are skin tested; a successful transfer is indicated by erythema and induration 24 hr later at the site of antigen injection. Although several workers have reported successful transfers, neither the phenomenon nor the molecule are understood (1). A major problem has been the lack of easily reproduced transfers of cellular immunity by immune cell extracts in animal systems (2-5).We have studied a dialyzable activity of guinea pig immune cell lysates by an in vitro correlate of cellular immunity, the macrophage migration inhibition test. In this test, immune peritoneal exudate cells incubated with specific antigen are inhibited from migrating when compared to immune cells incubated with no antigen or a nonspecific antigen. Nonimmune peritoneal exudate cells migrate the same distance in the presence and absence of antigen. Thus, one might test the ability of transfer factor to convert nonimmune cells to the immune state by monitoring the migration inhibition "responses" to antigen of nonimmune cells treated with transfer factor. Since we test the "transfer factor" exclusively in vitro, the molecule or molecules we study may not be the same as those defined as transfer factor in human systems.Fireman et al. (6) and Asher et al. (7) have tested human transfer factor by the blastogenesis test, an in vitro correlate of cellular immunity. Their results indicate that preparations of human transfer factor, active in vivo, may also be active in vitro.MATERIALS AND METHODS Preparation of Transfer Factor. Fourteen days after footpad injection of ovalbumin (OA) or bovine gamma globulin (BGG) in complete Freund's adjuvant (8), a cell suspension in 4.0 ml of Hanks' balanced salt solution (Gibco) was prepared from the draining lymph nodes of a Hartley guinea pig. The cells were lysed by 10 freeze-thaw cycles, and centrifuged at 700 X g for 20 min at 220. The resulting supernatant was centrifuged at 20,000 X g for 40 min at 40, and the final supernatant was lyophilized, redissolved...