Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k7 = 0.3 ms, k4 = 2.3 ms, k3 = 6.9 ms, k6 = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k4), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M1 to M2 transition. However, experimentally two different M intermediates are not resolved, and M2 and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M2) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M2) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm-1 and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O.