The possible interaction of a second lysine with the retinylidene Schiff base of bacteriorhodopsin (Lewis, A., Marcus, M. A., Ehrenberg, B. & Crespi, H. (1978) Proc. Natl. Acad. Sci. USA 75, 4642-4646) has been investigated by specific incorporation of 15N into the epsilon-amino groups of the lysine residues. Comparison of resonance Raman spectra of bacteriorhodopsin grown on 100%, 0%, and 50% labeled lysine demonstrates that 15N isotope effects on the Schiff base vibration can be accounted for by 15N labeling only at the Schiff base nitrogen. Our data also provide in situ confirmation of the linkage of the retinal chromophore with the epsilon-amino nitrogen of lysine.