The (31)P NMR resonance from the inner and outer leaflets of DMPC in unilamellar vesicle bilayers has been split by use of the slowly penetrating paramagnetic shift reagent, Pr(3+). The perturbing effect of subsequently added iminosulfurane transdermal penetration enhancers (TPEs) is to accelerate the collapse of this splitting, especially in the case of the bromo derivative 3. The aforementioned acceleration of the splitting is enhanced by the addition of 16 mol% cholesterol. Conversely, 33 mol% cholesterol appears to seal the bilayer to the effect of the TPEs--even when present at 20 mol%. These observations are consistent with the deep penetration of the TPEs into the DMPC bilayer, i.e., the perturbation of the bilayer is transmembrane and supports a model in which a subset of the bromo TPE derivative 3 is kinetically trapped in the bilayer. This feature leads to an enhanced residence time of 3 in the bilayer, and by extension to the skin, and therefore to an explanation for the markedly enhanced activity of the bromo TPE derivative relative to that of other halogenated derivatives in the series of iminosulfuranes studied.