The secondary metabolite amygdalin is a cyanogenic diglucoside that at high concentrations is associated with intense bitterness in seeds of the Rosaceae, including kernels of almond (Prunus dulcis (Mill.), syn. Prunus amygdalus D. A. Webb Batsch). Amygdalin is a glucoside of prunasin, itself a glucoside of R-mandelonitrile (a cyanohydrin). Here we report the isolation of an almond enzyme (UGT85A19) that stereo-selectively glucosylates R-mandelonitrile to produce prunasin. In a survey of developing kernels from seven bitter and 11 non-bitter genotypes with polyclonal antibody raised to UGT85A19, the enzyme was found to accumulate to higher levels in the bitter types in later development. This differential accumulation of UGT85A19 is associated with more than three-fold greater mandelonitrile glucosyltransferase activity in bitter kernels compared with non-bitter types, and transcriptional regulation was demonstrated using quantitative-PCR analysis. UGT85A19 and its encoding transcript were most concentrated in the testa (seed coat) of the kernel compared with the embryo, and prunasin and amygdalin were differentially compartmentalised in these tissues. Prunasin was confined to the testa and amygdalin was confined to the embryo. These results are consistent with the seed coat being an important site of synthesis of prunasin as a precursor of amygdalin accumulation in the kernel. The presence of UGT85A19 in the kernel and other tissues of both bitter and non-bitter types indicates that its expression is unlikely to be a control point for amygdalin accumulation and suggests additional roles for the enzyme in almond metabolism.