Abstract Clinical evidence suggests that synovium can add to adjacent articular cartilage damage, potentially contributing to the development of osteoarthritis (OA). Inflammation of the synovium (synovitis) is dependent on the type of injury sustained, the time after injury and concomitant changes in other joint tissues. To define the role of synovitis in OA development, there is a need for baseline measures that can reliably distinguish synovial inflammation from normal synovium both within and between joints. This study tested the hypothesis that normal synovium from distinct anatomical locations in young and adult sheep is homogeneous with respect to consistently low molecular expression of the inflammatory mediators - tumour necrosis factor alpha (TNF-α) and interleukins (IL) such as IL-1β, IL-1Ra, IL-6 and IL-8. Additionally, maturation will not influence the expression of these select inflammatory biomarkers. Samples of synovium from four anatomic locations (medial and lateral margins, suprapatellar pouch (patella region), posterior to the posterior cruciate ligament, from each joint of 5 adult and 4 immature animals were graded histologically or analyzed for mRNA expression of inflammatory cytokines. Histologically, no evidence of synovitis was noted although some variance in sub-intimal fibrosis was observed between sample locations in mature sheep. Molecular expression of all inflammatory mediators was low and homogeneously expressed at constitutive levels in all sample locations. These findings confirm the hypothesis that the normal sheep synovium is a homogeneous tissue throughout the joint and establishes the baseline expression levels for several pro-inflammatory mediators in both immature and mature sheep.