Myristoyl-CoA:proteinN-myristoyltransferase (Nmt) catalyses the covalent attachment of myristate to the N-terminal glycine of a small subset of cellular proteins produced during vegetative growth ofCandida albicans. nmt447Dis a mutantNMTallele encoding an enzyme with a Gly447? Asp substitution and reduced affinity for myristoyl-CoA. Among isogenicNMT/NMT, NMT/dnmtandnmtd/nmt447Dstrains, onlynmtd/nmt447Dcells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 . When switched from YPD/myristate to YPD alone, 60% of the organisms die within 4 h. Antibodies raised against the C-terminal eight residues ofSaccharomyces cerevisiaeArf1p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains.N-Myristoylation ofC. albicansADP-ribosylation factor (Arf) produced a change in its electrophoretic mobility during SDS-PAGE: the myristoylated species migrated more rapidly than the nonmyristoylated species. In anNMT/nmtd, strain, 100% of the Arf isN-myristoylated based on this mobility shift assay. When exponentially growingnmtd/nmt447Dcells were incubated at 24 in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, = 50% of total cellular Arf was nonmyristoylated. This finding suggests that = 50% reduction in ArfN-myristoylation is a biochemical marker of a growth-arrested cell. A similar conclusion was made after assaying isogenicS. cerevisiaestrains containing various combinations ofNMT1, nmt1-451D, ARF1, arf1d, ARF2andarf2dalleles and grown at 24-37 on YPD or YPD/myristate. Peptidomimetic inhibitors ofC. albicansNmt were synthesized based on the N-terminal sequence of anS. cerevisiaeArf. SC-59383 has an IC50of 1.45 + 0.08 M for purifiedC. albicansNmt and is 560-fold selective for the fungal compared to humanN-myristoyltransf erase. It had an EC50of 51 + 17 and 67 + 6 M, 24 and 48 h after a single administration of the drug to cultures ofC. albicans.The Arf gel mobility shift assay indicated that a single dose of 200 M produced a < 50% reduction in ArfN-myristoylation after 4 h, which is consistent with the fungistatic, but not fungicidal, activity. The effect on Nmt was specific: an enantiomer, SC-59840, had no inhibitory effect on purifiedC. albicansNmt (IC50> 1000 M), and 200 M of the compound produced no detectable reduction in ArfN-myristoylationin vivo.SC-58272, which is related to SC-59383, was a more potent inhibitorin vitro(IC500.056 + 001 M), but had no growth inhibitory activity and did not produce any detectable reduction in Arf N-myristoylation. These findings highlight the utility of the Arf protein gel mobility shift assay for demonstrating the mechanism-based antifungal activity of SC-59383, a selective inhibitor of C. albicans Nmt.