A comparative study of absorption spectroscopy at 100 K has been performed on three-dimensional crystals of bacteriorhodopsin extd. from a lipidic cubic phase and on native purple membrane. A modified microspectrophotometer has been designed which yields absorption data with a high signal-to-noise ratio and remarkable reproducibility. Excellent agreement of the absorption spectra of the three-dimensional crystals and the purple membrane is obsd. provided that a rigorous crystal-handling procedure is followed. This result supports the equivalence of the protein structure in both the cubic phase crystals and the native purple membrane. On the other hand, it is shown that dramatic deviations of the crystal spectrum can be induced by minor changes in the extn. method. Exposure to air at room temp. can lead within a short time to an irreversible dehydration manifested by a distinct species with an absorption max. at 500 nm. Exposure of the crystals to a buffer with lower ionic strength than the crystn. soln. produces a different spectral form with an absorption max. at 477 nm, which was assigned to a distorted protein conformation induced by osmotic stress. The extreme sensitivity of these crystals to exptl. conditions is relevant for x-ray structural studies, in particular as different exptl. treatments are implemented to trap the intermediates of the protein's photocycle. [on SciFinder (R)]