In the present study, the effect of concentration, osmolality and charge of x-ray contrast agents on their diffusion and equilibrium distribution across different zones of cartilage was investigated. Full-thickness cartilage discs (Ø = 8.5 mm, n = 3) were extracted from healthy equine femoral condyle (n = 2). The diffusion of four different contrast agent baths (Condition A: Visipaque 320 mg/ml, 290 mOsmol/kg; Condition B: Visipaque 320 mg/ml, 600 mOsmol/kg; Condition C: Visipaque 160 mg/ml, 290 mOsmol/kg; Condition D: Hexabrix 320 mg/ml, 600 mOsmol/kg) was allowed only through the articular surface. Samples were imaged with a micro computed tomography scanner (micro-CT) before the contrast agent bath was applied, and after 5, 10, 20, 30 minutes and 1, 2, 3, 4, 5, 6, 7, 10, 12, 24, 30, 36, and 48 hours. Findings show that osmolality and concentration do not have a pronounced effect on diffusion. However, concentration influence on diffusion is seen on zonal curves. Moreover, the diffusion coefficient of Hexabrix was between 2.9 and 8.6 times lower than that of Visipaque that reflects the important effect of solute’s charge on the transport through charged hydrated tissue such as articular cartilage. Slightly different diffusion coefficient observed within dilute and concentrated Visipaque baths suggested deviation from ideal Fickean behavior within articular cartilage. However, close diffusion coefficients of cartilage exposed to low and high osmolality baths confirmed the minor effect of osmolality on the transport of neutral solutes.