Leukocyte adherence mediated by intercellular adhesion molecule-1 (ICAM-1) binding to leukocyte function-associated antigen (LFA-1) is required for proper inflammatory and immune function. Inhibition of ICAM-1\LFA-1 binding using monoclonal antibodies (mAb) has been shown to be efficacious at inhibiting lymphoma metastasis as well as leukocyte emigration into tissue in a number of inflammatory diseases such as ischemia-reperfusion injury, septic shock and rheumatoid arthritis. In this report, we describe the development and characterization of a small peptide antagonist of ICAM-1-dependent cell aggregation. By using repeated selection of a cyclic nonapeptide phage display library on purified ICAM-1, we identified phage that were competitively eluted with anti-ICAM-1 mAb. The peptide sequences were determined by nucleotide sequencing, and the peptide sequence (C*LLRMRSIC*) (IP01) that occurred most frequently was chosen for further study. Phage expressing this peptide sequence specifically bound ICAM-1 over a range of 5 x 10(6) to 1 x 10(8) phage/microL. A cyclic IP01 peptide, linear IP01 peptide, a cyclic nonapeptide with a scrambled IP01 sequence, and a random, cyclic nonapeptide were synthesized. The cyclic and linear IP01 peptides were able to inhibit ICAM-1-mediated cell aggregation at a concentration of 1 mM, whereas the random and scrambled peptide sequences did not alter aggregation. Cyclic IP01 had a half-maximal inhibitory concentration of approximately 970 microM. Cyclic IP01 did not inhibit cellular aggregation that was dependent on ICAM-2 or ICAM-3. Alanine substitutions in the cyclic IP01 identified at least four amino acids necessary for inhibition of ICAM-1 dependent cell aggregation; leucine 2, leucine 3, methionine 5, and arginine 6. Finally, we showed that cyclic IP01 can inhibit firm adhesion of neutrophils to endothelium, a critical event in inflammatory diseases, in an assay that recapitulates physiologic flow conditions. Homology of IP01 with the primary amino acid sequences of the alpha or beta subunit of LFA-1 was not identified. Thus, we identified a unique molecule that inhibits ICAM-1 dependent cell adhesion, but is not related to the primary sequence of the ICAM-1 ligand LFA-1. Due to the small size and ability to block cell-cell adhesion, IP01 may serve as a useful tool for study of ICAM-1 and LFA-1 biology as well as for the development of small molecule therapeutics.