The purpose of this study is to examine the intracellular distribution of collagen types I, III and V in tenocytes using triple-label immunofluorescence staining technique in high-density tenocyte culture on Filter Well Inserts (FWI). The tenocytes were incubated for 4 weeks under monolayer conditions and for 3 weeks on FWI. At the end of the third week of high-density culture, we observed tenocyte aggregation followed by macromass cluster formation. Immunofluorescence labeling with anti-collagen type I antibody revealed that the presence of collagen type I was mostly around the nucleus. Type III collagen was more diffused in the cytoplasm. Type V collagen was detected in fibrillar and vesicular forms in the cytoplasm. We conclude that, the high-density culture on FWI is an appropriate method for the production of tenocytes without loosing specialized processes such as the synthesis of different collagen molecules. We consider that the high-density culture system is suitable for in vitro applications which affect tendon biology and will improve our understanding of the biological behavior of tenocytes in view of adequate matrix structure synthesis. Such high-density cultures may serve as a model system to provide sufficient quantities of tenocytes to prepare tenocyte-polymer constructs for tissue engineering applications in tendon repair.