It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples  Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate (rans-ABA and (rans-ABA glucose ester and that this accumulation is due to (rans-ABA biosynthesis. 18O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [18O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [18O]ABA with two 18O atoms in the carboxyl group. We further show, by feeding experiments with [2H6]ABA-alcohol and 18O2, that this doubly-carboxyl-labeled ABA is synthesized from [18O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [2H6]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Like-wise, carbon monoxide inhibits the synthesis of doubly-carboxyllabeled ABA in 18O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant.