Adult mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate along the osteogenic, chondrogenic or adipogenic lineage (1). The exact physiological function of MSCs remains unclear at present, however, MSCs do play a role in tissue healing (2). Therefore, MSCs represent a source of adult stem cells which could have immense potential for use in reparative medicine. The endocannabinoid system is comprised of the cannabinoid (CB) G-protein coupled receptors (CB1 and CB2), their endogenous ligands and degradative enzymes. The endocannabinoid system has recently been established as a major regulator of the musculoskeletal system (3). The aim of this study was to examine the role of the endocannabinoid system in the differentiation of MSCs along the osteogenic lineage. MSCs were isolated from the bone marrow of adult Wistar rats and expanded in culture. MSCs were treated with osteogenic factors (OFs; 10mM β-glycerolphosphate, 10nM dexamethasone, 50μM ascorbic acid) to induce osteogenesis. In some experiments MSCs were treated with the CB1 receptor antagonists, AM251 or SR141617A (both 1μM). MSCs were treated for 2 weeks, RNA extracted and osteocalcin mRNA and CB1 mRNA were measured by qPCR. Osteocalcin immunoreactivity was assessed by immunocytochemistry. Hydroxyapaptite deposits in the extracellular matrix were measured using a commercial kit following treatment of the cells with OFs, AM251 or SR141617A for 5 weeks. Osteocalcin mRNA was significantly increased from 0.3±0.1 (arbitrary units, mean±SEM) to 7.4±3.1 (p<0.01, unpaired t-test, n=3-6) by osteoinductive factors. Also, osteocalcin mRNA was significantly increased from 0.3±0.1 to 1.2±0.1 and 2.3±0.8 when MSCs were exposed to AM251 (1μM) and SR141617A (1μM), respectively (p<0.01 vs., control cells, unpaired t-test, n=6), indicative of osteogenic differentiation. Incubation with SR141617A in the presence of OFs further increased the expression of osteocalcin mRNA from 7.4±3.1 in cells treated with OFs alone to 15.7±5.1 (p<0.05, Newman-Keuls Multiple Comparison test, n=3-6). Hydroxyapaptite deposits in the extracellular matrix were significantly increased from 1.9±0.1 (arbitrary units, mean±SEM) to 5.9±0.6 by OFs and further significantly increased to 7.3±0.3 when MSCs were exposed to AM251 in the presence of OFs (p<0.05, unpaired t-test, 8 replicates). CB1 mRNA expression was significantly increased from 1.6±0.8 (arbitrary units, mean±SEM ) to 5.5±1.2 by OFs and further increased to 6.3±0.6 in MSCs treated with SR141617A (p<0.01, unpaired t-test, n=3-7). These data show that the cannabinoid receptor system is upregulated during MSC osteogenesis and that osteogenesis is enhanced following pharmacological blockage of the CB1 receptor.