Purpose: Glucosamine (GLN) products are taken by patients suffering from OA and other joint problems. In the United States it sold in the form of Glucosamine HCL (GHCl) as a nutritional supplement, while in Europe it is prescribed in the Glucosamine Sulfate (GS) form. In clinical trials both in the US and Europe has shown that GLN can reduce pain scores in patients with moderate to severe knee pain. Although GLN may be beneficial, their exact mechanism of action(s) remains obscure. Also because of the two different forms used, there is still controversy to which form of GLN is more effective. To further elucidate their mechanism of action, we determined the effects of both forms GLN at concentrations attainable in vivo and lower than those reported in in vitro studies, on selected pain mediators, inflammatory mediators, collagenases MMP-1 and MMP-13 using cytokine-stimulated human chondrocyte cultures. Methods: We took advantage of the recent pharmacokinetic information of GLN concentrations in plasma and synovial fluid to conduct our studies. Human (n=3) OA chondrocytes (ages: 53, 59 & 62) were isolated from cartilage obtained from total knee arthroplasty. Chondrocytes were seeded at 0.5 X 106 cells/ml and cultured in the presence of DMEM plus 10% FBS for 48h. Pharmaceutical grade GS and GHCl were provided as kind gifts from Bioiberica, SA, Spain. The chondrocytes were then serum starved for 24h and treated with either of the following: Control (Ctrl), 5 ng/ml human recombinant IL-β (IL-1), 0.5μg/ml GS, 5.0μg/ml GS, 0.5μg/ml GHCl, 5.0μg/ml GHCl, IL-1 + 0.5μg/ml GS, IL-1 + 5.0 μg/ml GS, IL-1 + 0.5μg/ml GHCl or IL-1 + 5.0 μg/ml GHCl. Cells and media were collected 48h after stimulation. Total RNA was isolated from cells. Semi-quantative RT-PCR was uses to assess gene expression for Kininogen, IL-1β, IL-1β receptor, MMP-1 and MMP-13. Proteins of MMP-1 and MMP-13 secreted into the media were measured by Western blot. Gene and Western blot data were analyzed statistically using the student t-test. Different letters within a parameter indicate significant differences (P <0.05) differences between treatments. Results: As expected, the addition of 5ng/ml of IL-1β stimulated the mRNA expression of IL-1β, IL-1 receptor, Kininogen, MMP-1 and MMP-13 in the OA chondrocytes. Chondrocytes cultured in the preseence of 0.5GS, 5.0 GS, 0.5 GHCl and 5.0 GHCl were all able to down-regulate the mRNA expression of these genes significantly. In most cases, both the GS and GHCl were able to repress expression to or below control levels. Western blotting of MMP-1 and MMP-13 confirmed gene expression data at the protein level. Both GS and GHCl decreased the protein abundance of cytokine stimulated MMP-1 and MMP-13. The addition of GS showed more potency as compared to GHCl at the same concentrations in suppressing the gene expression of these mediators. Conclusions: The most common symptom and complaint of OA patients is joint pain which often increases in severity until the only clinical option is total joint arthroplasty. Despite the central position of pain in the progression and management of OA, little is known about the causes of OA pain. Pain receptors are present in cartilage but the functional significance of their presence is not known. A possibility may be the generation of soluble pain mediators that migrate to adjacent tissues to elicit an effect. Pain mediators in cartilage may also play a role in cartilage degradation. Low grade inflammation occurs in OA resulting in joint pain. The addition of both forms of GLN was effective in depressing expression of Kininogen, IL-1β and its receptor. This would indicate that GLN can downregulate inflammatory effects such as further inflammation, pain and cartilage matrix turnover. The concentration of GLN at 0.5μg/ml is about 6 times lower than that found in human serum after GLN ingestion. More studies that investigate the signaling cascade involved in this regulation of pain mediators, inflammatory cytokines and MMPs will allow us to understand the molecular action of GLN in OA pain and as a treatment for OA.