NMT1 is an essential Saccharomyces cerevisiae gene which encodes myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). Nmt1p transfers myristate (C14:0), from myristoyl-CoA to the amino-terminal Gly residue of several essential cellular proteins. Little information is available about how myristoyl-CoA metabolism is regulated in eukaryotic cells. We have isolated and characterized three unlinked Fatty Acid Activation genes from S. cerevisiae, FAA1, FAA2, and FAA3. In vitro biochemical assays reveal that the myristoyl-CoA synthetase activity of purified Faa2p is approximately equal to that of Faa1p, and two orders of magnitude greater than that of Faa3p. Analysis of NMT1 strains containing faa1, faa2, and/or faa3 null alleles indicates that Faa1p, Faa2p, and Faa3p are not essential for vegetative growth when de novo acyl-CoA synthesis by fatty acid synthetase (Fas) is active. S. cerevisiae strains containing nmt1-181 exhibit temperature-sensitive growth arrest and myristic acid auxotrophy due to the reduced affinity of its mutant protein product (nmtGly451-->Asp) for myristoyl-CoA. Comparison of the growth characteristics of isogenic NMT1 and nmt1-181 strains with all possible combinations of faa1, faa2, and faa3 null alleles, in the presence or absence of an active Fas complex, indicates that (i) Faa1p is responsible for activation of imported fatty acids to their CoA derivatives; (ii) Faa2p and Faa3p are able to access endogenous but not imported fatty acid substrates; (iii) nmt181p requires myristoyl-CoA production from both Fas and Faas for cells to remain viable at nonpermissive temperatures; (iv) Faa2p is unique among the three Faas in its ability, when overproduced, to partially rescue growth of a nmt1-181 strain at nonpermissive temperatures on yeast/peptone/dextrose (YPD) media without C14:0 supplementation; (v) acyl-CoAs produced by Faa1p, Faa2p, or Faa3p are not specifically targeted for beta-oxidation; and (vi) the ability of NMT1, faa1 delta, faa2 delta, faa3 delta strains to remain viable in the absence of an active Fas complex on YPD plus C14:0, or on media that contains fatty acids as the sole carbon source, suggests that S. cerevisiae contains other acyl-CoA synthetases which can activate imported fatty acids.