Attenuated total reflection spectroscopy, where precise control of external parameters is feasible, was combined with the step-scan technique, which provides time-resolved Fourier transform-infrared spectra with microsecond time resolution. The advantages of this new approach were demonstrated by analyzing the photoreaction of the membrane protein bacteriorhodopsin (BR). By variation of temperature and pH clear-cut separation of the intermediate states L, M, N, and O, was achieved while previous infrared studies failed to separate the O intermediate in the wild-type, Therefore, we focused on a detailed description of the O-BR difference spectrum. It was proved that the major changes in secondary structure of BR are reversed in the N to O transition. Bands at 1432 and 1448 cm(-1) were tentatively assigned to CH3 deformation vibrations of the retinal chromophore In the O state. At 1713 cm(-1) the protonation of a carboxylic amino acid during the lifetime of O was observed. In addition to the wild-type data, the long-lived O intermediate of the E204Q and the E204T mutant have been investigated.