The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36°C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24°C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24°C. Complementation of growth at 36°C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451→Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent K(m) for myristoyl-CoA at 36°C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.