In this study we describe peripheral corneal endothelial cell migrationin vitroin the absence and presence of a ROCK-inhibitor. For this study, 21 corneal endothelial graft rims, with attached trabecular meshwork (TM), were prepared from Descemet membrane-endothelial cell sheets by 6.5 mm trepanation. For the initial proof-of-concept, 7 outer graft rims were cultured in a thermo-reversible hydrogel matrix for up to 47 days. To assess the effect of a ROCK-inhibitor, 14 paired outer rims were cultured either with or without ROCK-inhibitor for up to 46 days. At the end of culture, tissue was retrieved from the hydrogel matrix and examined for cell viability and expression of different endothelial cell markers (ZO-1, Na+/K+-ATPase, NCAM, glypican, and vimentin). All cultured rims remained viable and displayed either single regions (n = 5/21) or collective areas (n = 16/21) of cell migration, regardless of the presence or absence of ROCK-inhibition. Migration started after 4±2 days and continued for at least 29 days. The presence of ROCK-inhibitor seemed to contribute to a more regular cell morphology of migrating cells. In addition, 7 outer rims demonstrated a phenotypically distinct late-onset but fast-growing cell population emerging from the area close to the limbus. These cells emerged after 3 weeks of culture and appeared less differentiated compared to other areas of migration. Immunostaining showed that migrated cells maintained the expression patterns of endothelial cell markers. In conclusion, we observed 2 morphologically distinct migrating cell populations with the first type being triggered by a broken physical barrier, which disrupted contact inhibition and the second, late-onset type showing a higher proliferative capacity though appearing less differentiated. This cell subpopulation appeared to be mediated by stimuli other than loss of contact inhibition and ROCK-inhibitor presence. Further exploration of the differences between these cell types may assist in optimizing regenerative treatment options for endothelial diseases.