Bone marrow-derived human mesenchymal stem cells (hMSCs) lack the Coxsackie-adenovirus (Ad) receptor and thus are poorly transduced by vectors based on human Ad serotype 5 (Ad5). We investigated whether this problem could be overcome by using tropism-modified Ad5 vectors carrying fiber shaft domains and knobs of different human species B Ads (Ad5FBs). To allow quantitative analyses, these vectors coded for the enhanced green fluorescent protein (eGFP). Transgene expression analysis showed superior transduction of hMSCs by all Ad5FBs tested as compared with conventional Ad5 vectors. This was evident both by the frequency of eGFP-positive cells and by the eGFP level per cell. Highly efficient transduction of hMSCs, with limited variability between cells from different donors, was achieved with vectors displaying fiber domains of Ad serotypes 50, 35, and 16. These findings could not be reconciled with the very low levels of CD46, a recently identified receptor for species B Ads, on hMSCs, suggesting that AdFBs probably use receptors other than CD46 to enter these cells. We further observed that high eGFP levels were maintained in replication-restricted hMSCs for more than 30 days. In dividing hMSCs, foreign DNA delivered by Ad5FBs was expressed in a large fraction of the cells for approximately 3 weeks without compromising their replication capacity. Importantly, the transduced hMSCs retained their capacity to differentiate into adipocytes and osteoblasts when exposed to the appropriate stimuli.