N-myristoylation involves the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of a protein via a covalent amide bond. Two N-terminal glycine myristoyltransferases, NMT1 and NMT2, have been identified as enzymes for this reaction. NMT1 and NMT2 are well known to be specific for N-terminal glycine, and even mutating the glycine to a slightly larger alanine will disrupt myristoylation by NMT1 and NMT2. In rare instances like small GTPase protein ARF6, NMT1 and NMT2 can also transfer myristoyl to the ɛ-NH2 of lysine residues near the N-terminal. My thesis project began with investigating the substrates of APT2, an acyl-protein thioesterase, using biorthogonal chemical probes. Surprisingly, my study reveals that APT2, with an N-terminal cysteine, can be N-terminal myristoylated. Subsequent experiments showed that human NMT1 and NMT2 efficiently catalyze the myristoylation of N-terminal cysteines of APT1 and APT2. APT1 and APT2 are previously known to be S-palmitoylated on their N-terminal cysteines, which is important for their endomembrane localization. APT1 is reported to localize to mitochondria and Golgi, while APT2 is known to localize specifically to the Golgi. Inhibitors specific to NMTs, like DDD86481, effectively eliminate N-myristoylation and S-palmitoylation. Furthermore, these inhibitors abolish the localization of APT1 and APT2 on the endomembrane. This suggests that N-myristoylation is a crucial factor in regulating the localization of APTs to endomembranes. Our findings unveil a novel sequence motif for N-myristoylation and suggest that other substrates with N-terminal residues like cysteine, alanine, and serine may be regulated by this previously unrecognized function of NMT.