We studied the effects of co-culture with peritoneal macrophages stimulated by M-CSF in vivo or in vitro on mouse embryonic development in vitro by evaluating the rate of blastocyst formation from pronuclear stage embryos. Further, we measured several cytokines in the co-culture medium, and examined the effects of cytokines on mouse embryonic development to clarify the mechanism of the co-culture effects. An addition of recombinant human M-CSF in the culture of embryos alone significantly enhanced the embryonic development. Co-culture with peritoneal macrophages also significantly enhanced the embryonic development, and the addition of M-CSF in the co-culture showed an additve effect. Similarly, co-culture with peritoneal macrophages isolated from mice which were pretreated with an i.p.injection of M-CSF significantly augmented the embryonic development compared with the culture of embryo alone and the co-culture with untreated macrophages. Moreover, we found that an indirect contact with macrophages, i.e.the supernatant from the macrophage culture, made the same effect on embryonic development as a direct contact with macrophages in co-culturing. Several cytokines, such as interleukin (IL) -1b, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN) -g, were detectable on a considerable level in the co-culture medium, and they, except GM-CSF,appeared to be further produced by M-CSF stimulation, though an addition of these cytokines (200 pg/ml IL-1b, 20 ng/ml IL-6,100 pg/ml GM-CSF,or 50 pg/ml IFN-g) failed to make a positive effect on embryonic development. These results indicated that the co-culture with macrophages activated by M-CSF further improved mouse embryonic development, possibly mediated by soluble factors including cytokines secreted from macrophages.