The usefulness of stroboscopic time-resolved Fourier transform IR spectroscopy for studying the dynamics of biological systems is demonstrated. By using this technique, we have obtained broadband IR absorbance difference spectra after photolysis of bacteriorhodospin with a time resolution of approximately 50 microseconds, spectral resolution of 4 cm-1, and a detection limit of delta A less than or equal to 10(-4). These capabilities permit observation of detailed structural changes in individual residues as bacteriorhodopsin passes through its L, M, and N intermediate states near physiological temperatures. When combined with band assignments based on isotope labeling and site-directed mutagenesis, the stroboscopic Fourier transform IR difference spectra show that on the time scale of the L intermediate, Asp-96 has an altered environment that may be accompanied by change in its protonation state. On the time scale of the L----M transition, this Asp-96 perturbation/deprotonation is largely reversed, and Asp-85 becomes protonated. During the M----N transition, Asp-85 appears to remain protonated but undergoes a change in its environment as evidenced by a shift of vC = O from 1761 to 1755 cm-1. The retention of a proton on Asp-85 in the N state indicates that the proton transferred from the Schiff base to this residue in the L----M step is not released to the extracellular medium during the same photocycle, but rather during a subsequent one. Also during the M----N transition, Asp-96 undergoes a deprotonation (possibly for the second time in a single photocycle). Bands in the amide I and amide II spectral regions in the M----N difference spectrum indicate the occurrence of a conformational change involving one or more peptide groups in the protein backbone.