Partitioning of the small amphipathic spin labels 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-oxo-2,2,6,6-tetramethylpiperidinyl- N-oxy (TEMPONE), and di-tertiary butyl nitroxide (DTBN) into interdigitated L β I and non-interdigitated L β ′, P β ′, and L α phases was compared. The double chain interdigitated phases examined were 1,2-dipalmitoylphosphatidylcholine (DPPC) in the presence of 2.17 M ethanol, dihexadecylphosphatidylcholine (DHPC), and the complexes of dipalmitoylphosphatidylglycerol (DPPG) with polymyxin B and myelin basic protein. The triple chain mixed interdigitated phase of the asymmetric chain 1-stearoyl-2-caproyl-phosphatidylcholine (18:10-PC) was also examined. The non-interdigitated lipids were DPPC, DPPC in the presence of 0.87 M ethanol, DPPG, and the complex of DPPG with polylysine. Quantitation of the percentage of the membrane-bound spectral contribution in the presence of NiSO 4, used to broaden the aqueous spectral contribution, showed that all three spin labels partitioned into the L β I phase significantly more than into the L β ′ phase. The hyperfine splitting values indicated that all three spin labels were located near the apolar/polar interface in the L β I phase. The amount of TEMPO and DTBN bound to the L β I phase was similar to that in the L α phase but more TEMPONE was bound to the L β I phase than to the L α phase. This is attributed to the greater polarity of TEMPONE, its preference for a polar or amphipathic environment, and the larger volume between lipid head groups at the apolar/polar interface in the L β I phase compared to the other phases. However, TEMPO binding to the mixed interdigitated gel phase of 18:10-PC was no greater than to the non-interdigitated L β ′ phase of DPPC, reflecting the smaller volume at the apolar/polar interface of this triple chain interdigitated bilayer compared to the double chain interdigitated bilayers. Thus interdigitation of the lipid bilayer, whether due to addition of a substance like ethanol, or spontaneous as in DHPC, can cause increased partitioning of other substances into the bilayer, provided that they are small enough to fit into the volume created at the apolar/polar interface. This increased partitioning could contribute to an increased permeability of small molecules across interdigitated bilayers.