AbstractPreimplantation mouse embryos were cultured for 48 hours from the four‐cell and eight‐cell stage to the blastocyst stage in the presence of Cyclophosphamide (CPA) or one of its metabolites–4‐hydroperoxy‐CPA (4‐HP‐CPA), phosphoramide mustard (PAM), and acrolein (Acr)‐to identify the metabolite which is embryotoxic after CPA treatment of pregnant mice during the preimplantation period. The dose‐response relations for the inhibition of blas‐tulation revealed identical inhibition curves for PAM and 4‐HP‐CPA (in solution 4‐HP‐CPA immediately decomposes to 4‐hydroxy‐CPA (4‐OH‐CPA)). These two metabolites are inhibiting blastulation in vitro at concentrations that are 10,000 times lower than CPA and 100 times lower than acrolein. When blastocysts which had developed in the presence of CPA and its metabolites in vitro were subsequently cultured in inhibitor‐free medium NCTC‐109, the same dose‐response relationship pattern was obtained. Since 4‐OH‐CPA decomposes into acrolein and PAM in vivo and in vitro and since PAM and 4‐OH‐CPA exhibit identical embryotoxicity towards preimplantation embryos in vitro, PAM probably also is an active embryotoxic CPA metabolite in vivo before implantation. This result is discussed in relation to the importance of alkylating CPA metab‐olites in cancer treatmen