Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic expts. performed at room temp., controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form obsd. in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes contg. bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystn. and/or room temp. conditions as the main source of this heterogeneity. This assignment is supported by an expt. showing interconversion of the species upon intentional dehydration and by crystallog. data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystn. process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallog. data, due to its ability to det. quant. compositional heterogeneity resulting from proteins in different protonation states. [on SciFinder (R)]