We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABA_A receptor as a model system. The most common form of the GABA_A receptor probably consists of 2α₁-, 2β₂- and 1γ₂-subunits. We show here that the arrangement of subunits around the central Cl⁻ ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding α₁-, β₂- and γ₂-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [³H]-Ro15-1788, a specific radioligand for the GABA_A receptor. A His₆-tag on the α₁-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His₆ immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135°, close to the value of 144° expected if the two α-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABA_A receptor rosette, and can also be applied to other ionotropic receptors.