The influence of pH and temperature on the structural organization, fluidity and permeability of the hyperthermophilic archaeon membrane was investigated in situ by a combination of electron paramagnetic resonance (EPR) and fluorescence emission spectroscopy. For EPR measurements, Aeropyrum pernix cells, after growing at different pHs, were spin-labeled with the doxyl derivative of palmitic acid methylester (MeFASL(10,3)). From the EPR spectra maximal hyperfine splitting (2A (max)) and empirical correlation time (tau (emp)), which are related to mean membrane fluidity, were determined. The mean membrane fluidity increases with temperature and depends on the pH of the growth medium. Computer simulation of the EPR spectra shows that membrane of A. pernix is heterogeneous and consists of the regions characterized with three different types of motional characteristics, which define three types of membrane domains. Order parameter and proportion of the spin probes in the three types of domains define mean membrane fluidity. The fluidity changes of the membrane with pH and temperature correlate well with the ratio between the fluorescence emission intensity of the first and third bands in the vibronic spectra of pyrene, I(1)/I(3). At pH 7.0 a decrease of I(1)/I(3) from 2.0 to 1.2, due to the penetration of pyrene into the nonpolar membrane region, is achieved at temperatures above 65 degrees C, the lower temperature limit of A. pernix growth.