Previous (18)O labeling studies of abscisic acid (ABA) have shown that apple (Malus domestica Borkh. cv Granny Smith) fruits synthesize a majority of [(18)O]ABA with the label incorporated in the 1'-hydroxyl position and unlabeled in the carboxyl group (JAD Zeevaart, TG Heath, DA Gage [1989] Plant Physiol 91: 1594-1601). It was proposed that exchange of (18)O in the side chain with the medium occurred at an aldehyde intermediate stage of ABA biosynthesis. We have isolated ABA-aldehyde and 1'-4'-trans-ABA-diol (ABA-trans-diol) from (18)O-labeled apple fruit tissue and measured the extent and position of (18)O incorporation by tandem mass spectrometry. (18)O-Labeling patterns of ABA-aldehyde, ABA-trans-diol, and ABA indicate that ABA-aldehyde is a precursor to, and ABA-trans-diol a catabolite of, ABA. Exchange of (18)O in the carbonyl of ABA-aldehyde can be the cause of loss of (18)O from the side chain of [(18)O]ABA. Results of feeding experiments with deuterated substrates provide further support for the precursor-product relationship of ABA-aldehyde --> ABA --> ABA-trans-diol. The ABA-aldehyde and ABA-trans-diol contents of fruits and leaves were low, approximately 1 and 0.02 nanograms per gram fresh weight for ABA-aldehyde and ABA-trans-diol, respectively, while ABA levels in fruits ranged from 10 to 200 nanograms per gram fresh weight. ABA biosynthesis was about 10-fold lower in fruits than in leaves. In fruits, the majority of ABA was conjugated to beta-d-glucopyranosyl abscisate, whereas in leaves ABA was mainly hydroxylated to phaseic acid. Parallel pathways for ABA and trans-ABA biosynthesis and conjugation in fruits and leaves are proposed.