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We examined the formation of F-actin filaments and the expression of beta 1 integrin in human vascular endothelial cells cultured on type V collagen. The cells attached to the exogenous substrate, formed complete F-actin filaments, and expressed vinculin and beta 1 integrin 0.5-1 h after inoculation. These phenomena are referred to as the first ECM-integrin-cytoskeleton system. After 3-6 h, disassembly of the F-actin filaments was observed to occur from the leading edges, and the cells developed focal adhesions only in their central regions. After 12-24 h, the cells on the type V collagen failed to form the second ECM-integrin-cytoskeleton system, and gradually detached from the substrate. In contrast, the cells on type I collagen developed both the first and second system, and acclimatized themselves to the environment. Thrombospondin (TSP), an anti-adhesive protein, was capable of inhibiting the spreading of the cells both after 1 h and 24 h. However, type V collagen treated with TSP inhibited the cell spreading after 1 h, but not after 24 h. The attachment and spreading of the cells on type V collagen were little affected by an anti-TSP antibody and the synthetic peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), which significantly inhibited the attachment and spreading of the cells on TSP. Taken together, these results suggest that the cell detachment from type V collagen is not attributed to endogenously produced TSP (or member of TSP family) with anti-adhesive properties, but resulted from the failure to reconstitute the second ECM-integrin-cytoskeleton system in focal adhesion.