An alternative approach, represented by the present study, is to include in the ring units that improve the passive permeability of the MPs irrespective of the conformation of the peptide. We hypothesized that the permanent positive charge on the IP + heterocycle would attract IP + -containing MPs to a membrane, and that the hydrophobic nature of the ring system would facilitate movement across it. The PAMPA data shown in Figure strongly support this notion. 45% of the IP+-containing MPs analyzed displayed excellent passive permeability (-logPe < 6.0) despite the fact that their molecular weights ranged from 650-850 Daltons. Even very large macrocycles with molecular weights of > 1000 Daltons (MP18 and MP19) displayed moderate passive permeability (-logPe = 6.4 and 6.3, respectively). This is clearly due to an effect of the IP + unit, as demonstrated by a comparison of the membrane permeabilities of comparable peptides cyclized using different linkage strategies (Figure , yellow dots). Preliminary CAPA data also show that these IP + MPs readily access the cytosol of living cells (Figure ). Another interesting point is that the substitution on the IP + unit also influences permeability substantially (compare MP38-MP41 in the second row of Figure ). While we cannot rule out the possibility that this influences the conformation of the macrocycle, it seems more likely that this is a direct effect of the chemical and physical properties of the IP + moiety. For example, the hydrophobic, electron-donating piperidine ring in MP40 appears to promote permeability to a greater degree than fused aromatic rings (MP38, MP39 and MP41). It will be interesting in the future to thoroughly assess substitution effects on the permeability of otherwise identical IP + MPs.