The secondary structure of bacterio-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), was detd. by Raman spectroscopy. Addnl. CD measurements revealed only negligible conformational differences between BO in apomembranes and BR in purple membranes. Therefore, the secondary structure of BR was derived from the Raman data of BO. The protein conformation consists of 72-82% helixes, 2-11% b-strands, and 11-17% b-turns. Only about half of the helical structures correspond to aI-helixes, the other half possess non-aI-helical structures. According to the anal. of the Raman data, the derived secondary structure of BR was obtained with high reliability for all structure classes which can be distinguished by this method within the given uncertainty range. This is a remarkable difference from recently published secondary structural data derived from CD measurements where the helix content was reported to be 50-80%. The inherent exptl. and methodol. uncertainties of the CD-technique leading to such a range of variation are critically discussed in comparison to the method of Raman spectroscopy. The combined application of Raman and CD spectroscopy, as performed here, is demonstrated to be a substantial improvement in the secondary structure detn. of retinal-contg. membrane proteins. Evidently, some of the recently proposed structural models of BR with a b-strand content of >11% can be ruled out. [on SciFinder (R)]