A method employing stable isotope labeling and fast-atom bombardment (FAB) tandem mass spectrometry has been developed to directly assess events of biosynthesis and metabolism of arachidonic acid containing phospholipid molecular species by cells carried in culture. Mast cells, cultured with [(13)C]linoleic acid, converted this precursor into arachidonic acid which was then incorporated into cellular phospholipids. Over a 24 hour period, the extent of label enrichment in each arachidonate-containing phospholipid molecular species was monitored by using negative FAB ionization with selected reaction monitoring. Specific incorporation of [(13)C17] labeled arachidonate was determined from the ratio of the carboxylate anions at m/z 320 and 303, which correspond to [(13)C17]arachidonate and unlabeled arachidonate, respectively, produced by collision-induced dissociation of each specific molecular anion. The use of [(13)C]linoleic acid as a precursor of arachidonic acid avoids the problem of changing the endogenous pool size by directly adding labeled arachidonic acid. Measurement of the [(13)C17]label also avoids interferences from endogenous isobaric fatty acids that are naturally present at low levels.