Placental implantation involves highly regulated trophoblast invasion of the endometrial stroma. TGF is a known regulator of this process. This study examines the effect of TGF on extravillous cytotrophoblastic cell (EVCT) migration in cocultures of first-trimester human chorionic villus explants and primary human endometrial fibroblasts. Migration of EVCTs was followed by phase-contrast time-lapse micros-copy and was shown to highly depend on the endometrial fibroblast matrix. Interstitial EVCT invasion was also analyzed by confocal microscopy of fluorescently prelabeled trophoblasts and endometrial fibroblasts. As expected, addition of TGF led to inhibition of EVCT invasion of the endometrial cell layer. This inhibition was characterized by formation of compact EVCT stacks at migration fronts and displacement of endometrial fibroblasts. We tested the role of the RhoA/Rho-associated kinase (ROCK) pathway, a TGF-dependent pathway known to regulate cell migration. Interestingly, blocking ROCK with the chemical in-hibitor Y27632 had an effect opposite to TGF activation because it promoted superficial EVCT migration on the en-dometrial cell layer. These data suggest a role for ROCK in the TGF-dependent control of trophoblast migration. Furthermore , they indicate that even though ROCK signaling plays a role in human trophoblast cell invasion, EVCT migration can still occur in the absence of ROCK activity. (Endocrinology 149: 4475– 4485, 2008)