Current validation studies of FETAX (Frog Embryo Teratogenesis Assay—Xenopus) suggest that fewer than 15% of test compounds will prove to be false negatives or positives in this in vitro teratogenesis screening assay. We have developed a metabolic activation system employing uninduced rat liver microsomes to convert proteratogens, thus reducing the number of potential false negatives. Microsomes were prepared by homogenizing livers and centrifuing the homogenate first at 600 and then at 9000 × g avg. The supernatant from these centrifugations was then centrifuged twice at 120 000 × g avg. By not inducing the rat liver with Aroclor 1254, toxicity from Aroclor metabolites was avoided. Xenopus blastulae were exposed to different concentrations of cyclophosphamide together with the microsomes, generating system, and antibiotics for a period of 96 h. Activation reduced the 96-h LC50 by 4-fold from >11.0 to 2.8 mg/mL. The EC50 malformation was reduced 3.4-fold from 6.8 to 2 mg/mL. The severity of malformation was also increased by activation. Activation of cyclophosphamide also caused a decrease in growth. Uninduced rat liver microsomes can be used as an acceptable in vitro metabolic activation system for FETAX.