The promotion of spinal fusion using bone autografts is largely mediated by the osteoinductive potential of progenitors/mesenchymal stem cells (MSC) that reside in the marrow spaces of cancellous bone. Iliac crest is the common autograft donor site, but its use presents an increased risk for donor site pain, morbidity and infection. Degenerative bone samples harvested during facetectomy might provide an alternative viable source of osteoinductive autografts. In this study, we conducted an intra-individual comparison of the osteogenic potential of isolated low passage MSC from both sources. Iliac crest and degenerative facet joints were harvested from eight consecutive patients undergoing transforaminal lumbar interspinal fusion due to lumbar spinal stenosis. MSC were isolated by collagenase digestion, selected by plastic adherence and minimally expanded for downstream assays. Clonogenic and osteogenic potential was evaluated by colony formation assays in control and osteogenic culture medium. Osteogenic properties, including alkaline phosphatase (ALP) induction, matrix mineralization and type I collagen mRNA and protein expression were characterized using quantitative histochemical staining and reverse transcription PCR. Spontaneous adipogenesis was analysed by adipocyte enumeration and gene expression analysis of adipogenic markers. Average colony-forming efficiency in osteogenic medium was equal between iliac crest (38 +/- 12%) and facet joint (36 +/- 11%). Osteogenic potential at the clonal level was 55 +/- 26 and 68 +/- 17% for iliac crest and facet joint MSC, respectively. Clonogenic and osteogenic potential were significantly negatively associated with donor age. Osteogenic differentiation led to significant induction of ALP activity in iliac crest (sixfold) and facet joint (eightfold) MSC. Matrix mineralization quantified by Alizarin red staining was increased by osteogenic differentiation, yet similar between both MSC sources. Protein expression of type I collagen was enhanced during osteogenesis and significantly greater in iliac crest MSC. Correspondingly, COL1A2 mRNA expression was higher in osteogenically differentiated MSC from iliac crest. Adipocyte numbers showed significant differences between iliac crest (63 +/- 60) and facet joint (18 +/- 15) MSC under osteogenic conditions. Negative (GREM1) and positive (FABP4) adipogenic markers were not differentially expressed between sources. MSC from iliac crest and degenerative facet joints largely display similar clonogenic and osteogenic properties in vitro. Differences at the molecular level are not likely to impair the osteoinductive capacity of facet joint MSC. Bone autografts from facetectomy would be viable alternatives as bone autografts for intervertebral spinal fusion in lumbar spinal stenosis.