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Nucleoside triphosphate diphosphohydrolases (NTPDases; apyrases) (EC 3.6.1.5) hydrolyze di- and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad divalent cation (Mg(2+), Ca(2+)) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgi-localized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.