Stearoyl-acyl carrier protein Delta(9) desaturase (Delta9D) produces oleic acid, a nutritionally valuable fatty acid containing a cis double bond between C-9 and C-10. This multiprotein diiron enzyme complex reacts with stearoyl-acyl carrier protein, reduced [2Fe-2S] ferredoxin, and O(2) to complete the highly regiospecific and stereoselective desaturation reaction. Interactions with the acyl chain provide stability to the enzyme-substrate complex, give an energetic contribution to catalytic selectivity, and help to order the electron transfer, O(2) binding, and C-H bond cleavage steps of catalysis. Reactions with natural acyl chains indicate the involvement of a highly reactive diiron intermediate capable of oxidizing secondary C-H bonds (bond dissociation energy approximately 95 kcal/mol), but also capable of diagnostic O-atom transfer reactions with the appropriate substrate analogues. For soluble Delta9D, the natural reaction may initiate at the C-10 position, in contrast to the well-established initial reactivity of the membrane enzyme homologue stearoyl-coenzyme A (CoA) Delta(9) desaturase at the C-9 position.