This chapter discusses the functional significance of myristoyl moiety in N-myristoyl proteins (NMT). Myristoyl-CoA: protein N-myristoyltransferase catalyzes the cotranslational transfer of myristate from myristoyl-CoA to the amino-terminal Gly residue of a variety of eukaryotic cellular proteins and proteins encoded by enveloped and nonenveloped viruses. Cellular N-myristoyl proteins have diverse functions. They include protein kinases, kinase substrates, phosphoprotein phosphatases, other types of proteins involved in signal transduction cascades, such as the α subunits of heterotrimeric G proteins and adenosine diphosphate (ADP)-ribosylation factors, which participate in intracellular protein and vesicular transport. Myristate appears to be critical for mediating protein–protein and protein–membrane interactions required for expression of the biological activities of many N-myristoyl proteins. Therefore, NMT is a potential target for antiviral, antifungal, and antineoplastic. Several tools are available for assessing the role of the myris group in the function of an N-myristoyl protein. An example of such tools is a multiplasmid expression system, which allows production of nonmyristoylated and myristoylated forms of an NMT in Escherichia coli, an organism that lacks endogenous NMT activity.