Photophysical techniques have potential for the development of optical sensors in monitoring and controlling fermentors. In the particular case of acetone-butanol-ethanol (ABE) fermentation, carried out by bacteria of the speciesClostridium acetobutylicum, we have developed two studies based on fluorescence spectroscopy. First, we measured the intrinsic fluorescence of NADH related to bacteria metabolism, leading to a linear relationship between the NADH specific fluorescence and the specific rate of butyric acid production. At the same time, we have correlated enzymatic activities (acetate kinase, butyrate kinase, acetoacetate decarboxylase) with NADH specific fluorescence. Second, we studied the fluorescence polarization of extrinsic DPH (1,6-diphenyl-1,3,5-hexatriene) related to membrane fluidity. A simultaneous increase in both DPH anisotropy (order parameter increase) and butanol production is observed. Even though these results seem contradictory, because of the well-known fluidizing effect of butanol on lipids, they can be explained by a homeoviscous response ofC. acetobutylicum to the presence of butanol during fermentation. Thus the apparent changes in fluidity could be the result of the adaptative membrane alteration.