Acid-lability distinguishes plasmalogens from diacylglycerolipids; the presence of an acid labile lipid-like material in animal cells was key to the discovery of this class of lipids [12]. We have used thinlayer chromatography (TLC) combined with acid-treatment to demonstrate the presence of acid-labile lipids in C. difficile. A more precise tool in the study of microbial plasmalogens is high resolution mass spectrometry [5–7] which can distinguish plasmalogen molecular species from diacyl glycerolipids having essentially the same molecular mass. In this paper we show that C. difficile has both phospholipids and glycolipids that consist of diacyl and plasmalogen species. In addition to the previously reported phosphatidylglycerol (PG),we have found cardiolipin in the tetraacyl and plasmalogen forms, monohexosyldiradylglycerol (MHDRG), dihexosyldiradylglycerol (DHDRG) and two unusual glycosyldiradylglycerols. Plasmalogens were most abundant in the phospholipids.