The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the gamma-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist alpha-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effects on [125I]TID labeling of the AChR. The regions of the AChR alpha-subunit that incorporate [125I]TID were mapped by Staphylococcus aureus V8 protease digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the alpha-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology.