Factors produced by both the preimplantation embryo and the endometrium initiate events leading to proliferation of the surrounding maternal tissue and stimulation of angiogenesis (new blood vessel formation), processes critical for successful implantation. We have demonstrated in vitro angiogenic activity in serum-free mouse embryo conditioned media (SGI Abstract #15, 1993). Therefore, we initiated experiments to characterize the mouse embryo angiogenic factor(s). Two-cell embryos recovered from superovulated female mice were cultured in serum-free media. Every 24 h, approximately 25% of the embryos were removed from culture and assayed for in vitro angiogenic activity in a chemotaxis (cell migration) bioassay. Embryos and embryo conditioned media significantly (P<0.01) stimulated BALB/c3T3 cell migration compared to identical unconditioned media. Following the chemotaxis assay, the embryos were removed from the Boyden chambers and tested for viability (trypan blue dye exclusion). Viable embryos were fixed and stored at 4°C until immunocytochemistry. Platelet-derived growth factor (PDGF-AA), transforming growth factor (TGF-α), and leukemia inhibitory factor (LIF) were detected in embryos fixed on coverslips using avidin-biotin immunoperoxidase procedures. Addition of either PDGF-AA or TGF-α antibody to the remaining embryo conditioned media did NOT neutralize the chemoattractant activity, but was capable of neutralizing the stimulation of cell migration induced by either growth factor alone. LIF is not active in the chemotaxis assay. These experiments support our original hypothesis that the preimplantation mouse embryo produces a factor(s) critical to implantation and successful pregnancy. Demonstration of LIF, PDGF and TGF-α binding to preimplantation embryos supports earlier observations of expression of these growth factor transcripts in the preimplantation embryo as well as growth factor activity in embryo conditioned media (Haimovici & Anderson 1993). The novel chemoattractant activity we have observed does not appear to be LIF, PDGF or TGF-α.