In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms a further link to glutaric dialdehyde which immobilises the protein molecules. Cyt c is immobilised on Au(ll!) by reaction with N-acetylcystein and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Imaging by in situ STM in a 20 mM phosphate buffer electrolyte with a Au/AuOx reference electrode could then be achieved, Protein was identified as hemispherical features on the surface with close to molecular resolution and with a quite different character compared both to the bare metal surfaces and to metal surfaces with only linker molecules attached. No subunits or side chains were visible, but the protein exhibited a grained surface appearance, possibly caused by mobile subunits or immobilising agent. (C) 1997 Elsevier Science S.A.