The in vitro protein binding behavior of diclofenac sodium (sodium[o-(2,6-dichloroanilino)phenyl]acetate) in plasma and synovial fluid was investigated by equilibrium dialysis. The drug was highly protein bound (˜ 99.5%) and the extent of binding remained constant for drug concentrations of 2–10 μg/mL. Comparable results were obtained with human serum albumin solution (45 g/L) indicating that albumin is probably the responsible protein. The extent of binding remained relatively constant for drug concentrations of 0.25–10 μg/mL when albumin concentrations were >25 g/L. For albumin concentrations <10 g/L, the extent of binding tended to decrease with increased drug concentration. This concentration (10 g/L) is substantially lower than that usually observed in plasma or synovial fluid of arthritic patients. Curvature of the Scatchard plot indicated the existence of two classes of sites. Excellent results were obtained from fitting of the data according to two classes of sites (r2 >0.999). Parameter estimates (SEM) of the number of binding sites, n1 and n2, and the corresponding association constants, k1 and k2, were 2.26 (0.55), 10.20 (0.69), and 1.32 (0.54) × 105 M−1, and 3.71 (1.11) × 103 M−1, respectively. Simultaneous samples obtained from arthritic patients indicate considerably higher total protein and albumin concentrations in plasma compared with synovial fluid, but the albumin:total protein ratios were essentially the same. There was very little difference in plasma binding in arthritic patients compared with normal subjects. The extent of binding in synovial fluid samples was consistently lower than that for plasma samples (mean ± SD of 99.5 ± 0.2% versus 99.7 ± 0.1%, respectively). The difference in extent of binding between plasma and synovial fluid samples was rather small, but reached statistical significance.