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This study was aimed to evaluate the concentrations of interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 beta (IL-1β) in normal and osteoarthritic (OA) joints as well as the influence of joint location and arthrocentesis on these concentrations. Interleukin-1 receptor antagonist and IL-1β levels were determined in the synovial fluid (SF) of 18 normal and 18 OA joints. In all normal joints, arthrocentesis was repeated after 1 hour. No significant difference of SF IL-1ra and IL-1β levels between metacarpophalangeal/metatarsophalangeal, radiocarpal, and talocrural joints was observed. There was no significant change in SF IL-1ra and IL-1β levels between first and second arthrocentesis detectable. Synovial fluid IL-1ra and IL-1β levels were significantly increased in OA joints compared to normal joints. Synovial fluid WBC count and protein concentration were not significant different between normal and OA joints. Synovial fluid WBC count and protein concentration as well as IL-1ra and IL-1β concentration were positive correlated. The anatomic location of high motion joints seems to have no influence on SF IL-1ra and IL-1β levels. Arthrocentesis did not increase SF IL-1ra and IL-1β levels within 1 hour after joint puncture. Increased SF IL-1ra, IL-1β, and protein concentrations as well as WBC counts seem to be indicators of joint inflammation, but on their own are not allowing an exact differentiation between healthy and mild OA joints due to great value ranges and value overlap. Yet it has to be further investigated if in combination with other biomarkers, a clearer differentiation of pathologic processes in the joint can be made.