This study histologically and immunohistochemically demonstrated developmental changes in cell morphology and expression of type-X collagen in the attachment of the Achilles tendon to the calcaneus in the rat. Although the site of attachment in the mature rat showed a well organized, direct insertion that was composed of tendon, fibrocartilage, calcified fibrocartilage, and bone, this four-zone structure was not observed in the immature 1-week-old rat. Formation of fibrocartilage was observed at 2 weeks, together with the hypertrophy of chondrocytes and the appearance of the secondary center of ossification. Type-X collagen was not detected either in chondrocytes in the attachment area at 1 week or in hypertrophic chondrocytes at the attachment at 2 weeks. In the 3-week-old rat, the secondary center of ossification extended to the area of attachment and type-X collagen was detected both in cartilage spicules within the secondary center of ossification and in cells found at the attachment adjacent to the secondary center of ossification. A four-zone structure had been established by 6 weeks and remained through 20 weeks. After 6 weeks, type-X collagen was identified both in the attachment of the tendon and beneath the calcaneal fibrocartilage. Type-X collagen is produced by cells in transitional zones between calcified and noncalcified tissue, such as the interface between articular cartilage and subchondral bone. In these areas, the expression of this protein persists through maturity and is not transient.