Purpose: To compare synovial fluid (SF) biomarker levels in samples from patients with rheumatoid (RA), osteoarthritis (OA), and normal organ donors for prognostic/diagnostic purposes. Methods: Synovial fluid (SF) was collected from the knees of 45 (6 males and 39 females) OA, 22 (14 females and 8 males) RA patients and 20 (15 females and 5 males) normal organ donors. Eight biomarkers analyzed by ELISA methods were evaluated: interleukin (IL)-1, Il-6, Il-8, Il-11, leukemia inflammatory factor (LIF), cartilage oligomeric protein (COMP), osteocalcin, and osteogenic protein-1 (OP-1). Multivariate analysis assessed the effects of gender and disease activity: WOMAC scores for OA samples, and SF WBC, ESR, CRP for RA samples. Multivariant KruskalWallis and Mann-Whitney Tests were used; p< 0.05 was considered significant. Results: The mean (±SD) age was: 53±9 years for OA, 54±11 for RA, and 52±7 for donors. No gender differences were identified between markers. In RA SF, the levels of 4 out of 5 tested cytokines/chemokines were higher than in OA and normal SF. The most significant differences were found for Il-6 and Il-8, where Il-6 concentration was 2.5-fold higher than in OA, 2587.59±6393.69 vs. 1045.68±5451.13 og/ml, respectively (p< 0.005), and 8-fold higher than in normal, 2587.59±6393.69 vs. 339.06±906.28, respectively (P< 0.002). The levels of Il-8 were 8-fold higher than in OA, 6490.04±16550.74 vs 849.48 og/ml, respectively (P< 0.0005) and normal, 6490.04±16550.74 vs 788.69±837.705 og/ml, respectively (P< 0.028). The differences for Il-11 were not as substantial, especially between RA and OA samples, while it was a 5-fold difference between OA and normal SF, 654.11±1411.844 og/ml, respectively (P< 0.003). The value of Il-1 was significantly higher in normal than OA, 9.81±2.72 vs 8.56±7.06 og/ml, respectively (P< 0.004); in the RA, it was a tendency for a higher concentration of Il-1 than in OA and normal, but it did not reach significance. Surprisingly, concentration of LIF was higher in normal samples than in RA (P< 0.044) and OA (P< 0.001). The highest levels of OP-1 were found in normal SF (541.92±28.61 ng/ml), which were almost 5-fold higher than in OA (112.40±124.64) (P< 0.001) and more than 2-fold higher than in RA SF (202.25±149.13 ng/ml, P< 0.001). No differences were detected in the levels of COMP or osteocalcin between the experimental groups. Although the differences between active and inactive states of OA or RA were insignificant, for cytokines and OP-1 each state was statistically different from normal. SF from both OA and RA had higher biomarker levels than normals, regardless of disease state, though there was a trend of higher chemokine levels in less active RA, and higher Il-1, Il-6, and Il-8 levels in active OA. Conclusions: Our findings suggest that the levels of pathophysiologically important biomarkers in SF of patients with OA and RA differ according to the mechanisms that drive each disease. Thus, Il-1, Il-11, LIF and OP-1 appear to be significant for OA; while Il-6, Il-8, and OP-1 may have significance for RA. As previously observed in cartilage, a strong negative correlation between the levels of OP-1 and Il-6 family of chemokines was seen. Larger studies are necessary to develop a biomarker algorithm that would be of diagnostic/prognostic use.