The inhomogeneous and anisotropic mechanical properties and structural components of articular cartilage (artC) may cause complex acoustic properties in this important tissue. In this study, we used 50-MHz ultrasound (US) to measure in vitro the depth-dependence and anisotropy of the US speed of artC collected from the bovine patellae. The US speeds of 18 disk artC specimens sampled from 18 different patellae were measured in two orthogonal directions. One full-thickness layer (n=18) and three horizontal slices (n=18 x 3) with approximately equal thickness were prepared from each artC disk and measured with the US beam perpendicular to the artC surface. One full-thickness vertical slice (n=10) was measured at different depths with the US beam parallel to the artC surface. The measured US speeds of artC in the two orthogonal directions were significantly different (p < 0.001). The US speeds also significantly increased with the increase of tissue depth for both measurement directions (p < 0.001). The US speeds of artC from the superficial to deep regions were 1518 +/- 17 (mean + SD), 1532 +/- 26 and 1554 +/- 42 m/s for the US beam parallel to the artC surface, and 1574 +/- 29, 1621 +/- 34 and 1701 +/- 36 m/s for the beam perpendicular to the artC surface. The sound speed of the full-thickness layer was 1636 +/- 25 m/s. The results suggested that the depth-dependence and anisotropy of the US speed in artC should be taken into account when US is used for the artC measurement. It was also demonstrated in this study, using additional specimens (5 x 2), that the swelling of artC after detaching from its subchondral bone could cause the change of its sound speed.