Cryptococcus neoformans is a fungal pathogen that causes chronic- meningitis in 10% of patients with AIDS. Genetic and biochemical studies were conducted to determine whether myristoyl-CoA:protein N-myristoyl-transferase (Nmt) is a target for development of a new class of fungicidal drugs. A single copy of a conditional lethal C. neoformans NMT allele was introduced into the fungal genome by homologous recombination. The allele (nmt487D) produces temperature-sensitive myristic acid auxotrophy. This phenotype is due, in part, to under-myristoylation of a cellular ADP ribosylation factor (Arf) and can be rescued by forced expression of human Nmt. Two isogenic strains with identical growth kinetics at 35 °C were used to test the biological effects of an Nmt inhibitor. CPA8 contained a single copy of wild type C. neoformans NMT. HMC1 contained nmt487D plus 10 copies of human NMT. Since a single copy of nmt487D will not support growth at 35 °C, survival of HMC1 depends upon its human Nmt. ALYASKLS-NH2, an inhibitor derived from an Arf, was fully depeptidized: p-[(2-methyl-1-imidazol-1-yl)butyl]phenyl- acetyl was used to represent the GLYA tetrapeptide, whereas SKLS was replaced with a chiral tyrosino] scaffold. Kinetic studies revealed K(i (app)) values of 1.8 ± 1 and 9 ± 2.4 μM for purified fungal and human Nmts, respectively. The minimal inhibitory concentration of the compound was 2- fold lower for CPA8 compared with HMC1. A single dose of 100 μM produced a 5-fold greater inhibition of protein synthesis in CPAB versus HMC1. The strain specificity of these responses indicates that the fungicidal effect was Nmt-dependent. These two strains may be useful for screening chemical libraries for Nmt-based fungicidal compounds with relatively little activity against the human enzyme.