Purified halorhodopsin was isolated from Halobacterium halobium as previously described (Duschl, A. et al. (1988) J. Biol. Chem. 263, 17016-17022). Two purple bands were eluted from phenyl-Sepharose column, indicating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography/mass spectrometry we could identify palmitate (which is only a minor lipid component of archaeal cells) among lipids associated with purple fractions. Typically the palmitate content of the first eluted band was higher than that of the second, indicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present depending on the fraction analysed. Very little or no palmitate was released from denatured halorhodopsin. By adding palmitate to buffers used in the phenyl-Sepharose chromatography, only one sharp purple band was collected, corresponding to the less retained halorhodopsin fraction. Pentadecanoic fatty acid could also affect the halorhodopsin chromatography. Chromatography of halorhodopsin in the presence of beta-mercaptoethanol showed only one band, corresponding to the more retrained halorhodopsin form. The two halorhodopsin fractions had different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of decay of the absorbance at 570 nm upon illumination. By following the decay of the absorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pKa values of deprotonation.