In plants, changes in the levels of oleic acid (18:1), a major monounsaturated fatty acid (FA), results in the alteration of salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. This is evident in the Arabidopsis ssi2/fab2 mutant, which encodes a defective stearoyl-acyl carrier protein-desaturase (S-ACP-DES) and consequently accumulates high levels of stearic acid (18:0) and low levels of 18:1. In addition to SSI2, the Arabidopsis genome encodes six S-ACP-DES-like enzymes, the native expression levels of which are unable to compensate for a loss-of-function mutation in ssi2. The presence of low levels of 18:1 in the fab2 null mutant indicates that one or more S-ACP-DES isozymes contribute to the 18:1 pool. Biochemical assays show that in addition to SSI2, four other isozymes are capable of desaturating 18:0-ACP but with greatly reduced specific activities, which likely explains the inability of these SSI2 isozymes to substitute for a defective ssi2. Lines containing T-DNA insertions in S-ACP-DES1 and S-ACP-DES4 show that they are altered in their lipid profile but contain normal 18:1 levels. However, overexpression of the S-ACP-DES1 isoform in ssi2 plants results in restoration of 18:1 levels and thereby rescues all ssi2-associated phenotypes. Thus, high expression of a low specific activity S-ACP-DES is required to compensate for a mutation in ssi2. Transcript level of S-ACP-DES isoforms is reduced in high 18:1-containing plants. Enzyme activities of the desaturase isoforms in a 5-fold excess of 18:1-ACP show product inhibition of up to 73%. Together these data indicate that 18:1 levels are regulated at both transcriptional and post-translational levels.