The photoactivatable carbene precursor 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ( [125I]TID) was tested as a probe for labeling lipid-embedded segments of the proteins of pure membrane bound (Na+ + K+)-ATPase. The probe labeled the alpha-subunit (100 kDa), its major tryptic and chymotryptic fragments of 78 kDa, 58 kDa, and 46 kDa, and the beta-subunit (38 kDa) from within the lipid bilayer to nearly the same specific activity. The labeling was resistant to extensive proteolysis and the distribution of label among the proteolytic fragments and the two subunits was independent of a 47-fold variation in concentration of [125I]TID. The data show that several transmembrane segments are distributed along the sequence of the alpha-subunit and that also the beta-subunit traverses the bilayer. [125I]TID provides a more uniform labeling of the transmembrane segments of the alpha-subunit and beta-subunit than that obtained with other hydrophobic reagents. This will facilitate further studies of the primary structure and folding pattern of the Na+,K+-pump proteins in the membrane.