The photocrosslinked chemical array format is useful not merely for screening protein ligands, but also for gaining insight into structure-affinity relationships (SARs). By probing an array of 2000 natural products, containing 50 bleomycin (BLM) derivatives, with cell lysates that overexpress RFP-fused Shble protein, we successfully observed interactions between Shble protein and BLMs on the array. Among the BLM derivatives, those that had long C-terminal tails were found to bind strongly. The binding signal intensities observed on the chemical array correlated well with the association constants, which were determined by isothermal titration carolimetry (ITC) experiments (r(2)=0.663), showing that the on-chip results were not an artifact of ligand immobilization. In addition to the C-terminal tails, the propionamide moieties in pyrimidoblamic acid (PBA) also appeared to be important for binding. The contributions of the propionamide moieties of PBA to binding were further supported by the X-ray structure of the complex of Shble protein and BLM A(6). These results provide insight into the structural requirements for recognition of BLMs by Shble protein.