This study proposes a three-dimensional co-culturing system of mesenchymal stem cells (MSCs) and nucleus pulposus (NP) cells from New Zealand white male rabbits to differentiate MSCs into NP-like cells. The preferable ratio of MSCs to NP cells and the effects of mechanical stimulation were investigated without biochemical reagents. The preferable ratio was investigated without mechanical stimulation using five groups: Group I (MSC control); Group If (NP cell control); and Groups III, IV, and V, for which the ratios of NP cells to MSCs were 1:1, 1:2, and 2:1, respectively. During culture for 10 days without stimulation, the proliferation of MSCs did not increase after day 4. NP cells proliferated more when co-cultured as in Group V. However, the degree of differentiation of MSCs increased significantly in Group V. The differentiation of NP cells decreased gradually over time. When mechanical stimulation was applied to Groups I, II, and V, it contributed to the differentiation of MSCs into NP-like cells, as well as to that of NP cells, but did not contribute to the proliferation of either cell type. The contribution of mechanical stimulation to differentiation was also confirmed by RT-PCR. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.