High frequency scanning acoustic microscopy coupled with the Multi Layer Analysis (MLA) method was used to investigate the age-related changes occurring in subchondral bone and cartilage during rat skeletal maturation. Patellae of 6 immature and 7 mature rats were fixed in methanol, cut in the sagittal plane of the patella and imaged in methanol at 400 MHz (3 μm spatial resolution) using a scanning acoustic microscope (SAM 2000). Acoustic impedance (Z) images of each sample were constructed using the local measurement of the reflection coefficient. Ultrasound images revealed that calcified cartilage porosity decreased with age and the interface between hyaline and calcified cartilage became more regular. The mean impedance values of mature (5.2 ± 0.4 MRayl) and immature calcified cartilage and bone (4.9 ± 0.7 MRayl) were not significantly different. In 6 mature samples, two regions could be distinguished beneath hyaline cartilage: a region corresponding to calcified cartilage where the lower Z (4.6 ± 0.5 MRayl) may indicate a low mineralized transitional zone between hyaline cartilage and bone; and a region of higher reflection coefficient (Z = 5.4 ± 0.5 MRayl) and probably higher mineralization in the deeper bone. Current results indicate that quantitative acoustic microscopy may be a new means to investigate changes occurring in the structural organization and mechanical properties of calcified cartilage and bone during the development of diseases like arthritis and osteoarthritis.