N-terminal myristoylation is a cotranslational lipid modification common to many signaling proteins that often serves an integral role in the targeting and/or function of these proteins. Myristoylation is catalyzed by an enzyme activity, N-myristoyltransferase (NMT), which transfers myristic acid from myristoyl coenzyme A to the amino group of a protein's N-terminal glycine residue. While a single human NMT cDNA has been isolated and characterized (hNMT-1), biochemical evidence has indicated the presence of several distinct NMTs in vivo, often varying in either apparent molecular weight and/or subcellular distribution. We now report the cloning and characterization of a second, genetically distinct human NMT (hNMT-2), as well as the isolation of the respective mouse NMT homologue for each human enzyme. The mouse and human versions of each NMT are highly homologous, displaying greater than 95% amino acid sequence identity. Comparisons between the NMT-1 and NMT-2 proteins revealed reduced levels of sequence identity (76-77%), indicating that NMT-1 and NMT-2 comprise two distinct families of N-myristoyltransferases. Transient transfection of either the hNMT-1 or hNMT-2 cDNA into COS-7 cells resulted in the expression of high levels of NMT enzyme activity. Both hNMT-1 and hNMT-2 were found to myristoylate several commonly studied peptide substrates with similar, but distinguishable, relative selectivities. Western analysis revealed that while hNMT-2 appeared as a single 65-kDa protein in transfected COS-7 cells, hNMT-1 was processed to provide four distinct protein isoforms ranging from 49 to 68 kDa in size. Collectively, these studies demonstrate a heretofore unappreciated level of genetic complexity underlying the enzymology of N-terminal myristoylation and suggest that the specific inhibition or regulation of either NMT in vivo may in turn allow for the selective control of particular myristoylation-dependent cellular functions.