This study examined the question whether or not preimplantation mouse blastocysts can metabolize progesterone (P). When young (Day 4) and implanting (Day 5) blastocysts were cultured in supplemented Eagle's minimum essential medium containing 0.4 μM [³H]P, metabolism of P and formation of metabolites were noticed at 10 h of culture. The metabolites accumulated in medium as the culture continued to 118 h. Three of the four metabolite fractions were identified, by crystallization to constant sp. act., to be 5α-pregnane-3,20-dione and 3β-hydroxy-5α-pregnan-20-one (or allopregnanolone), accounting for 22 and 57% of radioactivity, respectively, and a small amount (1∽10%) of 3α-hydroxy-5α-pregnan-20-one. This suggests that both Δ⁴-5α-reductase and 3α- and 3β-hydroxysteroid dehydrogenase are active. Day 5 blastocysts were much more active than Day 4 blastocysts in P metabolism. It is suggested that the ability of blastocysts to metabolize P could produce the following effects in the adjacent endometrium: 1) a lessening of P effects; and consequently 2) a change in P-estrogen interaction; and 3) possible effects from the metabolites. These local effects of embryos on the endometrium may be important for embryonic development and implantation.