Two spin-labelled derivatives of the 5-(2-indolyl)-2,4-pentadienoyl class of inhibitors of the vacuolar ATPase have been synthesised and their EPR properties characterised in phospholipid membranes. One spin-labelled inhibitor is the amide derivative of pentadienic acid and 4-amino-TEMPO (INDOL6), and the other is the 3-hydroxymethyl-PROXYL ester (INDOL5). The response of the EPR spectra to the chain-melting transition of dimyristoyl phosphatidylcholine (DMPC) bilayers demonstrates that both derivatives incorporate in phospholipid membranes. The axially anisotropic EPR spectra of INDOL6 in fluid DMPC membranes indicate that the indolyl-pentadienoyl inhibitors intercalate between the lipid chains, in the membrane. INDOL5, designed to possess additional internal segmental mobility, exhibits more nearly isotropic motion of the spin-label moiety in fluid membranes than does INDOL6. The EPR characteristics of INDOL5 are therefore well suited to detecting specific ligand-protein interactions. Progressive saturation EPR experiments with polar and hydrophobic relaxation agents (aqueous Ni2+ and oxygen) show that the nitroxide group is buried in the membrane, with the indole moiety providing the anchor at the membrane polar-apolar interface. Rates of spin-label reduction by externally added ascorbate confirm this assignment. These two spin-labelled derivatives provide complementary EPR probes of the lipid environment (INDOL6), and of ligand-protein interactions (INDOL5), for this class of V-ATPase inhibitor.