Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study, we demonstrate that Y-27632, a specific inhibitor of Rho kinase (ROCK) activity, significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore, treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably, hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally, we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus, Y-27632 provides a means to “kick-start” slow-growing human pluripotent stem cells, especially after being thawed from frozen stocks. Together, these results argue that Y-27632 is a useful tool in overcoming obstacles to studies involving the cultivation of both hES cells and human iPS cells. Mol. Reprod. Dev. 76: 722–732, 2009. © 2009 Wiley-Liss, Inc.