Summary. Mice were induced to superovulate and 2-cell embryos were cultured in Whitten's medium with 10 mg bovine serum albumin/ml (WM) as control, Medium WM with 2\m=.\3,4\m=.\6,23\m=.\1 or 46\m=.\2\ g=m\ gplasmin/ml, Medium WM with 14\m=.\6,29\m=.\1 or 145\m=.\7 \ g = m \ g plasminogen/ml, Medium WM with 0\m=.\1,0\m=.\2, 1\m=.\1or 2\m=.\2 \g=m\gtrypsin/ml, Medium WM with 0\m=.\2,0\m=.\3, 1\m=.\6or 3\m=.\3 \g=m\gpronase/ml and Medium WM with 10% heat-treated bovine serum (HTBS). Proteolytic activities in the culture media were evaluated at the start of the culture period and 10 days later. Blastocyst formation was significantly reduced in cultures supplemented with pronase and in the two higher levels of trypsin when compared to that in Medium WM. More embryos developed to the blastocyst stage in Medium WM + 2\m=.\3 or 23\m=.\1\ g=m\ gplasmin/ml and Medium WM + 14\m=.\6\ g=m\ gplasminogen/ ml than in Medium WM (P < 0\m=.\05). The incidence of hatching was significantly greater in Medium WM than in all plasminogen-and plasmin-supplemented media except for Medium WM + 29\m=.\1 \g=m\gplasminogen/ml. Although not significantly different, hatching was lower in Medium WM and Medium WM + 0\m=.\1 \g=m\gtrypsin/ml when compared to Medium WM + HTBS. Similar numbers of embryos completed the hatching process in Media WM, WM + 0\m=.\1 or 0\ m=. \ 2\ g=m\ gtrypsin/ml and WM + 0\m=.\3 \ g = m \ g pronase/ml. Since dissolution of the zona pellucida occurred within 96 h for embryos cultured in Media WM + 1\ m=. \ 6 or 3\ m=. \ 3\ g=m\gpronase/ml and WM + 1\ m=. \ 1 or 2\ m=. \ 2\ g=m\gtrypsin/ ml, hatching could not be evaluated. The incidence of attachment and subsequent trophoblastic outgrowth was significantly greater in media with plasmin, plasminogen and HTBS than in pronase-and trypsin-supplemented media or in Medium WM alone. The results suggest that the enhancement in embryo development observed in pl asmi n\x=req-\ and plasminogen-supplemented media is due not only to the provision of protease but to an additional trophic effect as well. Simple medium supplemented with plasmin or plasminogen is as able to support in-vitro development as is medium supplemented with serum.