The stereochemistry of the hydrogen elimination that occurs during the formation of the Δ4- and Δ2′-double bonds of abscisic acid has been determined from the 14C/3H ratios in abscisic acid biosynthesized by avocado fruit from [2-14C,(2R)-2-3H1]-, [2-14C,(2S)-2-3H1]- and [2-14C,(5S)-5-3H1]-mevalonate. Setting the 14C/3H ratio at 3:3 for [2-14C,(2R)-2-3H1]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-14C,(2S)-2-3H1]mevalonate was 3:1.63. Removal of the 3′-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3′-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-14C,(2R)-2-3H1]-mevalonate and to 3:1.05 for [2-14C,(2S)-2-3H1]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one 3H atom at the 6′-methyl position of abscisic acid from the [(2R)-2-3H1]- and [(2S)-2-3H1]-mevalonate. The presence of some label at positions 3′ and 4 when [(2S)-2-3H1]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.