In order to analyze the involvement of growth factors in the implantation mechanism, we examined the direct effects of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) on trophoblast outgrowth of the mouse blastocyst in vitro. ICR mouse blastocysts were cultured for 4 days on a culture plate in medium containing EGF or TGF-alpha or conditioned medium obtained from cultured endometrial epithelial cells. Blastocysts were also co-cultured with endometrial epithelial cells. The trophoblast outgrowth of these cultured blastocysts was observed daily and the percentage of outgrowing embryos was calculated and analyzed statistically by the chi-squared test. Analysis for the specific binding of 125I-EGF in outgrown trophoblasts was carried out by autoradiography. The co-culture (days 3 and 4) and the presence of EGF (10 ng/ml, day 4), TGF-alpha (1 ng/ml, day 3; 10 ng/ml, days 2 and 3; 50 ng/ml, days 2-4) or conditioned medium (days 3 and 4) significantly stimulated the rate of trophoblast outgrowth. Preincubation of the conditioned medium with monoclonal anti-EGF or anti-TGF-alpha antibody suppressed the stimulatory effect of the conditioned medium on trophoblast outgrowth. The specific 125I-EGF binding in outgrown trophoblasts was demonstrated by autoradiography. These results suggest that EGF and TGF-alpha play an important role in the implantation process by directly stimulating trophoblast development.