Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.