Electron spin resonance (ESR) spectroscopy and spin label techniques have been used to study the effects of fumonisin B1 (FB1) and hydrolyzed fumonisin backbone (AP1) on the structural and dynamic properties of phosphatidylcholine membranes at the molecular level. Multilamellar liposomes consisting of dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (EYPC) were used. Six different nitroxide spin labels were used to determine what effects FB1 may impart on the ordering and mobility of lipids in membranes. The experimental results disclose the following: (1) In the fluid phase membrane, FB1 significantly increases the fluidities of n-doxylstearic acid (SA) spin labels (SL) attached to carbons 5 and 7, which disorders the alkyl chains and perturbs the surface region of the bilayer; by comparison, minimal effects were detected near the center of the bilayer. (2) In the gel phase, FB1 and AP1 imparts marked rigidifying effects on membrane fluidity, which enlarges the change in ordering on the phase transition even further. (3) FB1 also restricts the mobility of the (rigid) cholestane spin label. (4) A reduction in mobility of the tempo-stearate spin label suggests that the tricarballylic acid (TCA) moieties of FB1 might mimic the structure of polar headgroups in phospholipids. The present results may provide additional mechanisms to elucidate the toxicological activities of the fumonisins.