Objective - To determine effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding putative mediators of osteoarthritis in bovine cartilage explants cultured for 2 weeks. Sample Population - Articular cartilage explants harvested from carpal joints of 4 Holstein steers after slaughter. Procedures - Cartilage disks were treated as follows: fetal bovine serum only (control treatment), human recombinant interleukin (IL)-1β (50 ng/mL; IL-1 treatment), GLN (5 μg/mL) with addition of CS (20 μg/ mL; GLN-CS treatment), and human recombinant IL-1β (50 ng/mL) with addition of GLN and CS (IL-1-GLN-CS treatment). Media were analyzed for nitric oxide and prostaglandin E2 (PGE2) release. Explants were subjected to quantitative real-time PCR analysis; expressions of mRNA for inducible nitric oxide synthase, cyclooxygenase-2, microsomal prostaglandin E synthase 1, matrix metalloproteinase (MMP)-3 and -13, aggrecanase-1 and -2, tissue inhibitor of metalloproteinase (TIMP)-3, type II collagen, and aggrecan were assessed. Results - IL-1-GLN-CS and GLN-CS treatments decreased nitrite release, compared with IL-1 treatment; IL-1-GLN-CS treatment decreased IL1-induced PGE2 release. Expressions of inducible nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E synthase 1 mRNA were abrogated by GLN-CS and IL-1-GLN-CS treatments. Interleukin-1-induced mRNA expressions of proteolytic enzymes were diminished by IL-1-GLN-CS treatment. Compared with control treatment, GLN-CS treatment decreased MMP-3 and aggrecanase-2 mRNA expression. Transcripts of TIMP-3 were increased by IL-1-GLN-CS treatment, compared with IL-1 treatment. Genes encoding type II collagen and aggrecan on day 14 were upregulated by GLN-CS and IL-1-GLN-CS treatments, compared with control treatment. Conclusions and Clinical Relevance - Treatment with GLN and CS consistently downregulated mRNA expression for inflammatory mediators and matrix degrading enzymes while increasing TIMP-3 transcripts.