Mesenchymal stem cells (MSCs) are multipotent progenitor cells which give rise to specialised skeletal cells, including chondrocytes, osteocytes and adipocytes. Under the appropriate conditions, these cells can be manipulated in vitro for the development of new tissue such as cartilage. Thus, MSCs pose as an ideal cell source for use in skeletal tissue engineering. When isolated from a donor source, the cells retain their multipotent potential in vitro and can differentiate along a specific lineage, depending on the local environment in which they reside. This study focused on elucidating the stimuli that enhance MSC differentiation along the chondrogenic route. Hypoxic signalling through hypoxia inducible factor-1α (HIF-1α) has been shown to promote chondrogenesis [1] and non-hypoxic stimulation of HIF-1α is currently under investigation. The endogenous cannabinoid system has recently been implicated in skeletal physiology [2]. Thus, the link between cannabinoids and chondrogenesis was examined in this study, including their possible role as non-hypoxic regulators of HIF-1α. MSCs obtained from the femora and tibiae of 3 month old Wistar rats were cultured with chondrogenic growth factors (100nM dexamethasone, 100ng/ml, transforming growth factor-β, 50mM ascorbic acid-2-phosphate), Δ9-tetrahydrocannabinol (Δ9-THC, 5µM) or URB597 (1µM) for 3 weeks. URB597 inhibits the fatty acid amide hydrolase enzyme that degrades anandamide and functions in enhancing endocannabinoid tone. Cells were examined for proteoglycan deposition and expression of collagen II, as markers of chondrogenesis. Histological analysis of proteoglycans revealed a significant increase from 5.35±0.52 (mean±SEM, arbitrary units) in control cells to 10.03±1.37 in cells treated with chondrogenic growth factors for 3 weeks (P<0.05, ANOVA, n=6). A similar increase was observed from 4.58±0.34 in control cells to 6.24±0.80 in Δ9-THC treated cells and to 6.70±0.38 in URB597 treated cells (P<0.05, ANOVA, n=6). Immunocytochemistry has shown that 3 week treatment with chondrogenic growth factors, Δ9-THC or URB597 induced an increase in collagen II immunoreactivity (n=6). The HIF-1α inhibitor, Vitexin (20µM), downregulated HIF-1α expression and attenuated the expression of collagen II, as assessed by immunocytochemistry (n=6). In conclusion, this study has demonstrated a regulation of MSC chondrogenesis by cannabinoids, under normoxic conditions via a pathway that involves, in part, HIF-1α. Optimisation of the in vitro conditions for chondrogenic differentiation of MSCs will have a significant impact in treating musculoskeletal defects in the ever advancing field of skeletal tissue engineering.