The three-dimensional structure of a synthetic peptide corresponding to the putative transmembrane segment M3 (amino acid residues 277-301) of the α subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by means of two-dimensional1H-NMR spectroscopy in a chloroform/methanol (1:1) mixture containing 0.1 M LiClO4. Complete resonance assignment has been performed using double-quantum-filtered COSY (DQF-COSY), TOCSY and NOESY spectra. The spatial structure has been calculated using the Diana program on the basis of integrated intensities of NOESY spectra, HN-C(α)H and HC(α)-CβH spin-spin coupling constants. Residues 279-297 of M3 form a right-handed helix (root mean square deviation is 0.032 nm for backbone atoms and 0.088 nm for all heavy atoms). The conformations of the 17 side chains have been unambiguously determined. The obtained structure is in accord with the photolabeling pattern of the membrane nicotinic acetylcholine receptor inAChR) which suggests α-helical structure of M3 in the labeled portion [Blanton, M.P. and Cohen, J.B. (1994) Biochemistry 33, 2859-2872].