Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. These were used for site-directed spin-labeling to determine the location and assembly of the major coat protein incorporated in bilayer membranes of dioleoylphosphatidylcholine. The membrane location of the spin-labeled cysteine residues was studied with molecular oxygen and Ni2+ ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-saturation electron spin resonance (ESR) spectroscopy. The section of the protein around Thr36 is situated at the center of the membrane. Residue Thr46 is placed at the membrane surface in the phospholipid head group region with a short C-terminal section, including Ala49, extending into the aqueous phase. Residue Ala25 is then positioned consistently in the head group region of the apposing lipid monolayer leaflet. These positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydrophobic section (residues 25-46), and then drop abruptly in the aqueous phase (residue 49). Additionally, the strong immobilization and low oxygen accessibility of residue 25 are attributed to steric restriction at the hinge region between the transmembrane and N-terminal amphipathic helices. Sequence-specific modulations of the ESR parameters are also observed. Relatively low oxygen accessibilities in the hydrophobic region suggest intermolecular associations of the transmembrane helices, in agreement with saturation transfer ESR studies of the overall protein mobility. Relaxation enhancements additionally reveal a Ni2+ binding site in the N-terminal domain that is consistent with a surface orientation of the amphipathic helix.