This study provides a unique correlation between a molecular biological and biochemical analysis of the extracellular matrix (ECM) macromolecules in one half of 28 human Achilles tendons with an immunohistochemical study of the other. Both the insertion site and the mid-tendon were studied. The insertion (enthesis) is characterized by three distinctive fibrocartilages, two in the tendon (enthesial and sesamoid) and one on the heel bone (periosteal). Thus, its structure contrasts markedly with the fibrous character of the mid-tendon. RT-PCR analyses were performed on RNA extracted from mid-tendon and from the tendon fibrocartilages to investigate transcription of collagens and proteoglycans. Western blotting was also used to identify and characterize these macromolecules, and immunohistochemistry to localize their distribution. The results demonstrate striking differences in the ECM between the mid-tendon and its insertion. Types I, III, V and VI collagens, decorin, biglycan, fibromodulin and lumican were found in both the mid-tendon and the fibrocartilages, although their precise distribution often differed with site. mRNA for type II collagen was constantly present in the fibrocartilages, but it was only found in the mid-tendon of one specimen. The patterns of distribution for versican and aggrecan mRNA were complimentary - versican mRNA was present in the mid-tendon and absent from the fibrocartilages, while aggrecan mRNA was present in the fibrocartilages and absent from the mid-tendon. The range and distribution of ECM molecules detected in the Achilles tendon reflect the differing forces acting on it - the mid-tendon largely transmits tension and is characterized by molecules typical of fibrous tissues, but the fibrocartilages must also resist compression and thus contain, in addition, molecules typical of cartilage.