A paramagnetic analogue of cholesterol (called 25-doxyl-27-norcholesterol (CNO)), labeled near the w-end of the hydrophobic tail, was used to study interactions of cholesterol with filipin. We observed by electron microscopy that CNO- and cholesterol-filipin complexes are structurally equivalent. Two kinds of complexes were seen by ESR spectroscopy and electron microscopy, depending on the stoichiometric R ratio between the antibiotic and sterol. When R was high, an immobilized ESR spectrum appeared, showing strong imbrication between CNO and filipin. When R was nearer to unity, an exchange-broadened spectrum emerged, corresponding to a new phase that was very rich in CNO (a fast exchange between spins could occur by nearest contacts). CNO was easily displaced from its complex (i) by gradual addition of genuine cholesterol; and (ii) by an excess of phospholipids, owing to the very poor affinity of CNO (and cholesterol, by extension) for filipin in the lipidic phase. Almost no difference appeared between the ESR spectra of oriented samples, i.e. the probe showed no long-range order in any complex of CNO with filipin.