Phospholipase D is an important enzyme in signal transduction in neuro
nal tissue. A variety of assays have been used to measure phospholipas
e D activity in vitro. The most typical measure of phospholipase D act
ivity is the production of phosphatidylethanol in the presence of etha
nol. Phosphatidylethanol is a product of transphosphatidylation activi
ty that is considered a unique property of phospholipase D. To support
transphosphatidylation activity, high concentrations of ethanol may b
e required. Furthermore, most assays in the literature utilize a deter
gent. These extreme conditions, detergent and ethanol, may alter phosp
holipase D and hinder the study of its regulation. In this manuscript
we describe an assay that eliminates these potentially confounding con
ditions. It utilizes high specific activity [H-3]butanol as a nucleoph
ilic receptor. This eliminates the need for high concentrations of alc
ohol. The substrate is an analog of phosphatidylcholine that contains
short-chain fatty acids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine. P
hospholipase D readily hydrolyzes this substrate in the absence of det
ergent. This novel assay should be useful in the further characterizat
ion of phospholipase D.