The aim of the present study was to evaluate the effect of three different techniques for isolation of human bone-marrow-derived mesenchymal stem cells (MSCs) on the in vitro differentiation of these cells. Furthermore, the use of BMP-2 was assessed and the effect of gene transfer of the BMP-2 gene into these cells was evaluated. Reverse transcription polymerase chain reaction showed that isolation of MSCs by outgrowth from cancellous chips resulted in expression of both osteogenic and chondrogenic markers, while cells cultivated from marrow aspirates exhibited only osteogenic markers. Intramuscular reimplantation of BMP-2-transfected cells in poly-(L/DL 70/30) lactic acid fleeces resulted in formation of mineralized tissue after 8 weeks in athymic rats, while control-transfected cells exhibited matrix synthesis without mineralized tissue after intramuscular reimplantation.