Few studies have analyzed the effect of stretching after immobilization on the structural and biochemical properties of tendons. Here, the effect of stretching and immobilization on the proximal (p), intermediate (i), and distal (d) regions of the deep digital flexor tendon in rats was analyzed. The d region was subjected to compression and tension forces, the i region was subjected to compressive forces and the p region received tension forces. Rats were separated into five groups: GI--control for GII; GII--immobilized rats; GIII--control for GIV and GV groups; GIV--immobilized and stretched rats; and GV--immobilized rats which were allowed free cage activity. GII showed a higher molecular organization in the d and p regions as detected by measuring optical retardation, a lower concentration of hydroxyproline in the i region and a significant decrease in noncollagenous proteins found in the three regions of the tendon. Regarding the glycosaminoglycans, diminishing dermatan sulfate and the absence of chondroitin sulfate in the i region were observed in GII when compared to GI. However, in the same region of GIV, higher concentrations of chondroitin and dermatan sulfate were observed along with a strong metachromasy. An increase in hydroxyproline content in the i region and a higher molecular organization in the d and p regions were observed in GIV. Apparently, the active isoforms of metalloproteinase-2 also increased after stretching in all regions. These results suggest that stretching after immobilization contributed to the increase in molecular organization and to the synthesis of extracellular matrix components.5329-38