An automated optical biosensor instrument for measuring molecular interactions (Pharmacia BIAcore) has been used to characterise the epitopes recognised by 15 monoclonal antibodies raised against recombinant human granulocyte colony-stimulating factor (G-CSF). The BIAcore combines an autosampler and integrated microfluidic cartridge for the introduction and transportation of samples to the sensor chip surface, with surface plasmon resonance to detect binding events. A rabbit anti-mouse Fc antibody, coupled to the sensor surface in situ using conventional protein chemistry techniques, was used to capture an anti-G-CSF monoclonal antibody. G-CSF was bound to this antibody by injection over the sensor surface. Multi-site binding experiments were then performed in which other anti-G-CSF monoclonal antibodies were injected sequentially over the surface, and their ability to bind to the G-CSF in a multimolecular complex monitored in real time. Results obtained using the biosensor have been compared with data obtained by cross competition studies using biotinylated antibodies or antibody binding studies using chemically or enzymatically derived G-CSF peptide fragments or synthetic peptides. The results of these studies are in excellent agreement with the data from the BIAcore, although modification of the antibody or G-CSF occasionally altered the epitope affinity.