Protein myristoylation occurs when the 14 carbon fatty acid, myristic acid, is covalently attached by amide linkage to a protein's N -terminal glycine by an N -terminal myristoyltransferase (NMT). A variation of this called heterogeneous acylation occurs in vivo only in retina when specific proteins are modified by myristic acid (14:0), tetradecenoic acid (14:1 n–9), tetradecadienoic acid (14:2n –6), and lauric acid (12:0). Myristic and lauric acids are relatively rare, comprising approximately 1% of the fatty acids in the retina. The unsaturated fatty acids 14:1 n–9 and 14:2 n–6 are less abundant, but can be synthesized in retina by retroconversion of 18:1 n–9 and 18:2 n–6 fatty acids, respectively. A previous quantitative study of acyl-CoA pools in bovine retina, heart, and liver found comparable levels of acyl-CoAs in each tissue, indicating that heterogeneous acylation is not due to limiting amounts of myristoyl-CoA in retina. In this current study the authors have characterized a panel of purified recombinant Type I and II NMTs found in retina and liver by assessing their utilization of the four acyl-CoAs used in vivo to acylate retina proteins. Acceptor peptides used in these assays were derived from the N -termini of src which is only myristoylated in vivo, and the cAMP dependent kinase A catalytic subunit which is heterogeneously acylated in retina, but myristoylated in other tissues. The authors have tested the ability of unlabelled acyl-CoAs to compete with [3H] myristoyl-CoA transfer, the efficacy of an NMT inhibitory protein (NIP71), and acyl-CoA affinity chromatography was used to isolate endogenous NMT inhibitory factor(s) from bovine heart and retina tissue homogenates. These results provide a basis of kinetic parameters and enzymatic characterization for Type I and Type II NMTs with two acceptor peptides and the four physiologically relevant fatty acid-CoAs found on retinal proteins, but do not indicate that heterogeneous acylation is a specialized function of any of the enzymes tested in this study.