The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH ( Δ29DHODH) and the DHODH from ( DHODH) with ubiquinone (Q ) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q . Q is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q . We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q is mediated by enzyme penetration. We can also show that DHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than Δ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q , as well as lipid composition, on enzyme binding.