The resumption of DNA synthesis in delayed implanting mouse embryos undergoing metabolic activation in vitro was examined. Blastocysts were recovered from ovariectomized mice, incubated for various intervals in basal Eagle's medium, exposed to 3H-thymidine, and prepared for light microscopic autoradiography. Following incubation the proportion of labeled cells increased from 4% at 1 hr to 30% by 24 hr. This increase in labeling was not uniform in all regions of the blastocyst, i.e., labeling was initially highest over the inner cell mass (ICM) but remained low over the polar and proximal mural trophoblast for 6 and 12 hr, respectively, and then began to increase. This pattern in the resumption of DNA synthesis during activation in vitro is similar to that reported in vivo (Given and Weitlauf, '81) and suggests that the mechanism responsible in intrinsic to the blastocyst rather than being a differential response to the intrauterine milieu. Furthermore, it appears that the ICM may play an essential role in the resumption of synthesis in the surrounding trophoblast.