Protein Nα-terminal acetylation (NTA) catalyzed by Nα-acetyltransferases (NAT) is one of the most common protein modifications in eukaryotes, occurring on approximately 50-70% of yeast and 80-90% of human soluble proteins. NAT has been shown to play a critical role in the functioning of protein such as stability, interaction and targeting. Recently, Nα-acetyltransferase 60 (Naa60 or NatF) has been identified in higher eukaryotes. In human Naa60 is reported to be anchored to the Golgi and responsible for NTA of both cytosolic and membrane proteins. In plant, however, the localization of Naa60 has not been thoroughly addressed. Therefore, this study foccuses on the subcellular localization of Arabidopsis Naa60. Computational analysis of the Naa60 amino acid sequence using PredictProtein reveals potential membrane localization with two amphipathic α-helices at the C-terminus. Transient expression of Arabidopsis Naa60 fused with EYFP at the N- and C- ends in tobacco leaf confirms its localization to the plasma membrane. Co-expression with the RFP plasma membrane marker suggests that Naa60 also localize to the tonoplast.