Analysis of the mechanical properties of articular cartilage necessitates determination of thickness of the tested tissue. To evaluate the suitability of different methods for thickness measurements, the thickness of bovine and canine knee articular cartilage was determined with optical (stereomicroscopic), needle probe and ultrasonic techniques. The results obtained with the stereomicroscope and the needle probe showed high, linear correlations (r = 0.97, n = 80). The mean thickness obtained with the needle was slightly higher than the optical thickness (0.88 +/- 0.36 mm vs 0.85 +/- 0.34 mm, mean +/- S.D., n = 80, p < 0.01, matched-pairs Student t-test) or the ultrasonic thickness (0.93 +/- 0.42 mm vs 0.87 +/- 0.36 mm, n = 45, p < 0.05). The high scatter between optical and ultrasonic thickness, considered to be due to complex measurement geometry of canine knee articular cartilage, invalidated the use of the A-mode, 10 MHz-ultrasonic device for thickness measurements. Based on the results of uncertainty analysis it is concluded that optical and needle probe methods can be used interchangeably when determining shear modulus of articular cartilage with indentation tests. However, if high area-aspect ratios (indenter radius-to-cartilage thickness ratios) are used in the indentation measurements uncertainty in shear modulus may be markedly increased due to possible errors in the measurement of cartilage thickness.