Intrasynovial and extrasynovial flexor tendon grafts recently have been shown to have dissimilar patterns of cellular survival and host integration within the digital sheath. In an effort to determine if fundamental differences exist between these two types of tendons, we investigated the biochemical composition and cellular activity of intrasynovial and extrasynovial tendon segments of 12 adult mongrel dogs in short-term explant culture in MCDB 105 and in DMEM media. Proteoglycan, collagen and noncollagen protein synthesis and content and DNA synthesis were determined following culture in both media. Intrasynovial tendon segments cultured in MCDB 105 medium synthesized significantly less collagen, noncollagen protein, and DNA and had similar amounts of proteoglycans compared to extrasynovial tendons. Comparison of intrasynovial and extrasynovial tendon segment responses in DMEM medium showed that intrasynovial tendons synthesized more proteoglycan, protein, and DNA than they did in MCDB 105. Extrasynovial tendons had similar rates of matrix component and DNA synthesis in both media. Findings that the synthesis of matrix components and DNA between intrasynovial flexor and extrasynovial peroneal tendon segments differ significantly indicate that intrasynovial flexor tendons may be specially adapted to the nutritional milieu provided by an intrasynovial environment. These data are supported by the selective successful stimulation of fibrocartilaginous segments within intrasynovial flexor tendons in media favored for the culture of cartilaginous tissue.