Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Delta6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Delta9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Delta6 or Delta9 positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Delta6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Delta9-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Delta9-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Delta9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.