Almost all unsaturated fatty acids of biological origin contain only cis double bonds. Beacuse of this, an aditional enzyme is required: enoyl-CoA isomerase, that converts the Δ³-cis-acyl-CoA formed during these fatty acids degradation to a Δ²-trans-acyl-CoA. Such compounds are normal substrates of enoyl-CoA hydratase so that b oxidation can the continue. Since this there's no need of forming a double bond by dehydrogenation, there's no FADH2 prodution by acyl-CoA dehydrogenase, and so the energy produced is smaller.

Additional double bonds occur at three carbon intervals and are therefore never conjugated. Double bonds at these positions in fatty acids pose two problems for the b-oxidation pathway that are solved through the actions of three additional enzymes. The first problem is the formation of a Δ³-cis-acyl-CoA, and as said is solved by the action of enoyl-CoA isomerase.

The next problem is that the presence of a double bond at an even-numbered carbon atom results in the formation of 2,4-dienoyl-CoA, wich is a poor substrate for enoyl-CoA hydratase. However, NADPH-dependent 2,4-dienoyl-CoA reductase reduces the Δ⁴ double bond. The mammalian reductase yields trans-3-enoyl-CoA, wich, to proceed along the b-oxidation pathway, must first be isomerized by trans-2-enoyl-CoA isomerase.