Two novel cardiolipin derivatives were recently detected in Halobacterium salinarum, namely an archaeal analog of bisphosphatidylglycerol (BPG) and a glycocardiolipin (GlyC). GlyC was found to be tightly bound to bacteriorhodopsin. To obtain information on the presence and distribution of these archaeal cardiolipins, we have analyzed the lipids extracted from the crystallizer ponds of the salterns of Margherita di Savoia (Italy) and Eilat (Israel) and from cultures of representative species of the Halobacteriaceae by electrospray ionization mass spectrometry. BPG was present as a minor lipid component in the lipids extracted from the biomass of the Margherita di Savoia and the Eilat salterns, while GlyC was detected only in the extract of the biomass of Margherita di Savoia. Both compounds were enriched in the membrane fraction obtained by dialysis of the cells against distilled water. We detected BPG in all members of the Halobacteriaceae tested, but GlyC has so far been found only in the genera Halobacterium and Haloarcula. A sulfated diglycosyl diether was the major glycolipid detected in the biomass of both salterns.