Atomic force microscopy (AFM) was used to measure the forces stabilizing human aquaporin-1 (hAQP1), a tetrameric transmembrane protein that forms highly specific water channels. To this end, the AFM tip was attached to the C-terminus of hAQP1 and secondary structure elements were extracted from the membrane while the single-molecule force-extension curve was being recorded. Force peaks, reflecting the unfolding of secondary structure elements, could be interpreted in depth using the atomic model of hAQP1. Different classes of force-extension curves indicated the existence of alternative unfolding pathways for individual proteins. In addition, transmembrane helices at the periphery of the hAQP1 tetramer exhibited smaller extraction forces than helices at the interface between hAQP1 monomers. These results represent the first direct assessment of intermolecular forces defining the oligomeric state of a membrane protein. (C) 2003 Elsevier Science (USA). All rights reserved.