The platelet-reactive collagen III-derived fragment alpha1(III)CB4 has been synthesized as seven overlapping peptides, each as a homotrimeric triple-helical species covalently linked at the C terminus. Additional Gly-Pro-Hyp triplets were introduced at each end of the peptide sequence to ensure a stable triple-helical conformation at 20 degrees C, the temperature at which cell reactivity was measured. A Cys-containing triplet was included at each end to allow intermolecular cross-linking. All seven peptides in triple-helical, cross-linked form were able to cause platelet aggregation. Peptide 6, the most reactive species, was more aggregatory than collagen fibers. Platelet adhesion occurred to all peptides immobilized on plastic in monomeric form. Adhesion was integrin alpha2beta1-independent except in the case of peptide 6, adhesion to which was partially reduced by anti-integrin alpha2beta1 monoclonal antibodies. The presence of an alpha2beta1 recognition site in peptide 6 was confirmed using HT 1080 cells, which express alpha2beta1 as their major or sole collagen receptor. HT 1080 adhesion to both peptide 6 and collagen was strongly inhibited by anti-integrin alpha2beta1 monoclonal antibodies. These cells did not adhere to any of the other peptides. Comparison of the structure of peptide 6 with that of adjacent peptides indicates that the sequence Gly-Gly-Pro-Hyp-Gly-Pro-Arg, residues 522-528 of the collagen alpha1(III) chain, represents the minimum structure required for the recognition of alpha2beta1. Our findings support the view that the collagen triple helix possesses an intrinsic platelet reactivity that can be expressed independently of integrin alpha2beta1 and the precise level of which is governed by the exact nature of the primary sequence. Sequences such as those recognizing alpha2beta1 may potentiate the activity, whereas others may have the opposite effect.