High-resolution electron cryomicroscopy has clarified the tertiary structure of the proton-pumping bacteriorhodopsin (BR) at a resolution of 3.5 Angstrom in the direction parallel to the membrane. However, due to the lower resolution in the perpendicular direction, it could not be determined whether the retinal side-chain methyls at C-9 and C-13 were directed toward the extracellular surface or the cytoplasmic side of the membrane. The direction of the Me groups is of fundamental importance because it is directly related to the mechanisms of proton pumping. Our past bioorganic studies with C-9 substituents containing cc-sulfate groups suggested that the methyls are directed to the exterior. In contrast, measurements of linear dichroism, neutron diffraction and solid-state deuterium NMR have concluded the opposite. In order to secure further evidence, two tritiated analogs of retinal (5 and 6), each with a photolabile phenyl azide moiety appended through a 13-Angstrom spacer arm, one pointing in the same direction and the other pointing opposite to the polyene methyl groups, were synthesized and incorporated into the apoprotein to reconstitute functional bacteriorhodopsin analogs. The cross-linked residues labeled by analog 5, in which the photoactive group is directed opposite to the methyl groups, were identified as Arg-175 and Asn-176 by Edman degradation of cyanogen bromide-cleaved peptides. These two amino acids are located on the cytoplasmic side of alpha-helix F, indicating that the 9-Me in this photolabile analog points toward the extracellular space. These studies also revealed that the chromophore plane is closely perpendicular to the plane of the membrane and that the beta-ionone ring of retinal is tilted toward the extracellular space at a 27 degrees angle relative to the plane of the membrane. Photoaffinity studies with analog 6 did not give conclusive results.