Diheptanoyl phosphatidylcholine (DHPC) was reported to reduce inward sodium current in squid giant axons (Hendry et al., Biophys. J., 47, (1985) 841) and to decrease the frequency of the nicotinic acetylcholine-activated ion channel (Braun and Haydon, Pfügers Arch., 62, (1991) 418). To understand the DHPC effects, an influence of DHPC to increase dynamics/disorder (perturbation effect) in liposomes prepared from rat brain total lipids was studied at the 5th, 12th and 16th carbon membrane depths using the method of EPR spectroscopy of the spin labelled stearic acids and 1-palmitoyl-2-stearoyl phosphatidylcholines. The perturbation efficiency of DHPC in the liposomes was quantitated with the initial slope value of the order parameter S or inner splitting Amin versus DHPC concentration. DHPC at the DHPC/total lipid molar ratio of 1:10, 1:5, 1:3 and 1:2 increased dynamics/disorder of the liposomes. When the perturbation effect, compared at temperature scale, was normalized to 1 at the 5th carbon depth, the relative perturbation effect of DHPC was 1, 4.2, and 6.9 at the 5th, 12th and the 16th carbon depth, respectively, as detected by spin labelled stearic acids. Using the spin labelled lipids the perturbation effect was 1, 1.9 and 2.3, respectively. The differences of the perturbation effect of DHPC at different membrane depths correspond to the published perturbation effect of the local anesthetics lidocaine, tetracaine, dibucaine, heptacaine, IR-9 and carbisocaine on total lipid liposomes. The comparable perturbation properties of DHPC and of the local anesthetics support the hypothesis that the membrane perturbation caused by DHPC may play an important role in its effect on membrane function.