Integrin-mediated cell adhesion is central to cell survival, differentiation and motility. Many cell responses induced by integrins require both receptor occupancy and receptor aggregation, and appear to be regulated by both biochemical and biophysical means. Multidomain extracellular matrix molecules may serve to foster integrin aggregation by presenting local clusters of adhesion ligands, a hypothesis supported by studies with synthetic substrates showing that cell adhesion and migration are enhanced when adhesion ligands are presented in nanoscale clusters. Here, we used a novel synthetic polymer system to present the adhesion ligand GRGDSPK in nanoscale clusters with 1.7, 3.6 or 5.4 peptides per cluster against a non-adhesive background, where the peptide is mobile on a 2 nm polyethylene oxide tether. Average ligand density ranged from 190 to 5270 RGD/microm(2). We used these substrates to study the effects of ligand density and clustering on adhesion of wild-type NR6 fibroblasts, which express alphavbeta3 and alpha5beta1, integrins known to bind to linear RGD peptides. The strength of cell-substratum adhesion was quantified using a centrifugal detachment assay to assess the relative number of cells remaining adherent after a 10 minute application of defined distraction force. An unusual relationship between cell detachment and distraction force at relatively low values of applied force was found on substrates presenting the clustered ligand. Although a monotonic decrease in the number of cells remaining attached would be expected with increasing force on all substrates, we instead observed a peak (adhesion reinforcement) in this profile for certain ligand conditions. On substrates presenting clustered ligands, the fraction of cells remaining attached increased as the distraction force was increased to between 70 and 150 pN/cell, then decreased for higher forces. This phenomenon was only observed on substrates presenting higher ligand cluster sizes (n=3.6 or n=5.4) and was more pronounced at higher ligand densities. Adhesion reinforcement was not observed on fibronectin-coated surfaces. These results support previous studies showing that biophysical cues such as ligand spatial arrangement and extracellular matrix rigidity are central to the governance of cell responses to the external environment.