The dissection of the activities mediated by type I collagen requires an approach by which the influence of triple-helical conformation can be evaluated. The α1β1 and α2β1 integrin binding sites within type I collagen are dependent upon triple-helical conformation and contained within residues 124-822 from α1(I). Seven α1(I)-derived triple-helical peptides (THPs) were synthesized based on charge clustering (α1(I)256-270, α1(I)383- 396, α1(I)406-417, α1(I)415-423, α1(I)448-456, α1(I)496-507, and α1(I)526-537). Three additional THPs were synthesized (α1(I)85-96, α1(I)433-441, and α1(I)772-786) based on previously described or proposed activities (Kleinman, H. K., McGoodwin, E. B., Martin, G. R., Klebe, R. J., Fietzek, P. P., and Wooley, D. E. (1978) J. Biol. Chem. 253, 5642-5646; Staatz, W. D., Fok, K. F., Zutter, M. M., Adams, S. P., Rodriguez, B. A., and Santoro, S. A. (1991) J. Biol. Chem. 266, 7363-7387; San Antonio, J. D., Lander, A.D., Karnovsky, M. J., and Slayter, H. S. (1994) J. Cell Biol. 125, 1179-1188). Of the ten THPs, α1(I)772786 THP had the greatest activity, with half-maximal normal dermal fibroblast adhesion occurring at a peptide concentration of 1.6 μM. Triple-helicity was essential for activity of this sequence, as the non-triple-helical peptide analog (α1(I)772-786 SSP) exhibited considerably lower levels of cell adhesion promotion even at peptide concentrations as high as 100 μM. Within the sequence itself, residues 784-786 (Gly-Leu-Hyp) were important for cellular recognition, as the α1(I)772-783 THP had greatly reduced cell adhesion activity compared with α1(I)772-786 THP. Preliminary studies indicate that the β1 integrin subunit mediates fibroblast adhesion to α1(I)772-786 THP. The identification of fibroblast integrin binding sites within type I collagen may have important implications for understanding collagen metabolism.