Bone remodeling and, therefore, integration ofimplant materials require the coordinated regulation of osteo-blast and osteoclast activity. This is why thein vitroevalua-tion of biomaterials for bone regeneration should involve notonly the analysis of osteoblast differentiation but also the for-mation and differentiation of osteoclasts. In the presentstudy, we applied a material made of mineralized collagen Ithat mimics extracellular bone matrix to establish a culturesystem, which allows the cocultivation of human monocytesand human mesenchymal stem cells (hMSCs), which weredifferentiated into osteoclast-like cells and osteoblasts,respectively. Both cell types were cultivated on membrane-like structures from mineralized collagen. Transwell insertswere used to spatially separate the cell types but allowedexchange of soluble factors. The osteoclastogenesis andosteogenic differentiation were evaluated by analysis of geneexpression, determination of alkaline phosphatase (ALP), andtartrate-resistant acidic phosphatase (TRAP) activity. Further-more, cell morphology was studied using scanning electronand transmission electron microscopy. Osteogenicallyinduced hMSC showed an increased specific ALP activity aswell as increased gene expression of gene coding for alkalinephosphatase (ALPL), when cocultivated with differentiatingosteoclasts. Adipogenic differentiation of hMSCs was sup-pressed by the presence of osteoclasts as indicated by amajor decrease in adipocyte cell number and a decrease ingene expression of adipogenic markers. The formation ofmultinucleated osteoclasts seems to be decreased in thepresence of osteogenically induced hMSC as indicated byelectron microscopic evaluation and determination of TRAPactivity. However, gene expression of osteoclast markers wasnot decreased in coculture with osteogenically inducedhMSC.