The physiological significance of estradiol-17β for the early embryonic development in the pig was investigated in vitro by four different experimental designs. A total of 1635 morphologically intact morulae were cultured in vitro in Krebs-Ringer bicarbonate solution supplemented with 10% heat-inactivated lamb serum, and the blastocyst formation rate (BFR) was recorded after 24 or 48 h. The addition of estradiol-17β (0.1 nM, 1 nM, 100 nM), progesterone (100 nM, 500 nM) or cortisol (100 nM) to the culture medium did not affect BFR (95 to 100%, Experiment 1). Similarly, adding charcoal-stripped lamb serum to the medium instead of normal lamb serum in the absence or presence of 1 nM estradiol-17β had no effect (93 to 95% BFR, Experiment 2). The antiestrogen Nafoxidine, however, at a concentration of 15 μg/ml, significantly (p<0.01) reduced BFR to 13.3 ± 5.8% compared to controls (93.3 ± 4.2%, Experiment 3). Supplementation with estradiol-17β (1 nM) in the presence of 15 μg/ml Nafoxidine significantly (p<0.01) improved BFR to 57.2 ± 8.9%. Higher concentrations of estradiol-17β (100 nM, 100 μM) did not further enhance BFR. The stimulatory effects of estradiol-17β were specific since the BFR remained low in the presence of 100 nM progesterone (10.0 ± 4.5%) or 100 nM cortisol (3.3 ± 3.3%). Addition of 5% estradiol-17β-antiserum to the culture medium (Experiment 4) significantly (p<0.01) reduced BRF to 51.9 ± 6.7% compared to controls (93.1 ± 2.2%). Supplementation with 100 μM estradiol- 17β in the presence of 5% estradioi-17β antiserum significantly (p<0.05) improved BFR (79.7 ± 4.7%). It is concluded that estradioi-17β is an essential factor for the transformation of the compacted morula to the cavitated biastocyst stage. Our observations suggest that this effect is mediated through specific binding sites for estrogens.