Time-resolved, flash-induced difference absorbance spectra (300-700 nm) at pH 10.5 and 5 degrees C for the bacteriorhodopsin photocycle fast and slow decaying forms of the M intermediate (M(f) and M(s), respectively) and R intermediate are reported. The main distinguishing features are as follows: For M(f), DeltaA(max) = 412 nm, a shoulder at 436 nm, no absorbance change at 350 nm; DeltaA(min) = 565 nm; DeltaA(412)/DeltaA(565) = 0.85. For M(s), DeltaA(max) = 412 nm, a shoulder at 386 nm; DeltaA(min) = 575 nm; DeltaA(412)/DeltaA(575) = 0.6. For R, DeltaA(max) = 336 and 350 nm (double peak), smaller peaks at 386 and 412 nm; DeltaA(min) = 585 nm; DeltaA(350)/DeltaA(585) = 0.2. The different difference spectra for M(f) and M(s) provide direct evidence that these species, initially identified by their kinetics, are physically distinct. With fast transient absorption spectroscopy, it was shown that R may form very fast, perhaps faster than the L intermediate decays. On the basis of the different bleaching peaks for M(f) and M(s), we propose that M(f) and M(s) are in independent photocycles formed from slightly different forms of bacteriorhodopsin. R may also be in a different photocycle. The different forms of bacteriorhodopsin are probably in dynamic equilibrium with their ratios, controlled by pH and temperature.