We investigated the effects of cellular senescence on unidirectional endothelialization in vitro, simulating the anastomotic endothelialization of vascular prosthesis. The experiments were carried out with three different cumulative population-doubling levels (CPDLs) of bovine aortic endothelial cells (ECs), which have finite life span. Young ECs with 22 CPDL, middle aged with 46, and senescent with 70 at the time of inoculation were used. The effect of aging on unidirectional endothelialization, as well as cellular morphology and proliferative and migratory potentials of isolated cells, were qualitatively and quantitatively analyzed. The unidirectional endothelialization rate was determined by our newly designed method to prepare the square monolayer sheet with linear margins between cell-adhesion and noncell-adhesion regions. The results showed that endothelial cell senescence retarded not only proliferation and migration but also unidirectional endothelialization. Time-lapsed videomicroscopic study of unidirectional endothelialization process revealed that ECs at several rows back from the leading edge represented much slower rate of migration than did the ECs at the leading edge. These findings suggest that high cellular mobility observed for the ECs at the leading edge may result in localized excessive cell replication. Thus, atherosclerotic vessels containing senescent or injured ECs may have limited capability of anastomotic endothelialization.