The present report illustrates the response to osmotic stress of an extreme halophilic archaeon, Halorubrum sp., isolated from the saltern ponds of Margherita di Savoia in southern Italy. The hypotonic stress induces relevant changes in the membrane lipid composition: archaeal cardiolipin content markedly increases, whereas phosphatidylglycerol (PG) decreases. Membranes isolated from this archaeon after cell disruption by osmotic shock are highly enriched in archaeal cardiolipin and reveal the presence of a novel phospholipid. Electrospray ionization mass spectrometry and NMR analyses revealed that this novel lipid has the structure of a sulfo-diglyco-diether-phosphatidic acid, i.e., a phospholipid dimer or a novel cardiolipin analogue. As NMR analyses showed that the sugars in the novel phospholipid dimer are the same and in the same order of a sulfated diglycosyl diphytanylglycerol diether (S-DGD-5) present as a major lipid component in the archaeon membranes, the novel phospholipid dimer was named S-DGD-5-PA. We conclude that osmotic shock induces a specific increase in the membrane content of the two cardiolipins and suggest that PG and S-DGD-5 are intermediates for the de novo synthesis of archaeal cardiolipin and S-DGD-5-PA, respectively.