The identity of the lysine residue in bacteriorhodopsin to which the chromophore, retinal, is attached as a Schiff's base has been reinvestigated. Retinal is now shown to be linked to lysine-216 and not lysine-41, as had been concluded previously. The retinal in purple membrane was replaced by [15-3H]retinal, the Schiff's base linkage was reduced with NaBH4 while the sample was illuminated, and the resulting [retinyl-3H]bacterio-opsin was cleaved with CNBr. The radioactivity was present exclusively in the COOH-terminal peptide (amino acids 210--248). Sequence analysis showed that the [3H]retinal was attached to lysine-216. The same site was labeled when purple membrane was reduced with NaBH4 in the light at pH greater than 10 or at pH 8 and when membranes modified with ether or solubilized with hexadecyltrimethylammonium bromide were reduced in the dark. Our finding places of Schiff's base linkage close to the midpoint of the putative membrane-spanning alpha-helix that is directly connected to the COOH terminus of bacteriorhodopsin.