Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-L-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-D-serine (D-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of D-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence Of D-PS. Free D-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the Occurrence of a divalent cation-dependent enzyme that synthesizes D-PS by a base-exchange reaction. (C) 2009 Elsevier Inc. All rights reserved.