AbstractPhenylketophosphamide and phenylketoisophosphamide are preactivated acyclic ketone analogs of cyclophosphamide and isophosphamide with antitumor activity. These compounds undergo an elimination reaction to yield phosphoramide or isophosphoramide mustard and phenyl vinyl ketone. In this study, the embryotoxicity of phenylketophosphamide, phenylketoisophosphamide, and phenyl vinyl ketone were determined. Embryotoxicity was assessed in vitro in whole rat embryos cultured on day 10.5 of gestation in the absence and presence of an activating system derived from maternal liver. Both phenylketophosphamide and phenylketoisophosphamide were embryotoxic in the absence of metabolic activation. Moreover, there was no enhancement of this embryotoxicity in the presence of an activating system. A 10‐μM concentration of phenylketophosphamide produced 100% malformed embryos, while this concentration of phenylketoisophosphamide was not teratogenic. At 25 μM phenylketoisophosphamide, all the surviving exposed embryos were malformed. Phenylketophosphamide was embryolethal to more than 50% of the exposed embryos at a concentration of 50 μM. In contrast, a concentration of phenylketoisophosphamide of 100 μM was required to produce significant embryolethality. Phenyl vinyl ketone was not embryotoxic at any of the concentrations tested. The major malformation observed, a hypoplastic prosencephalon, and the growth retardation effects were not only similar for phenylketophosphamide and phenylketoisophosphamide, but also similar to those previously reported for “activated” cyclophosphamide. Unlike the results with cyclophosphamide, where both phosphoramide mustard and the aldehydic metabolite of cyclophosphamide, acrolein, are toxic, the embryotoxic effects of pheynlektophosphamide and phenylketoisophosphamide are mediated only by the mustard m