Arachidonic acid is selectively released during signal transduction in many cell types. To examine the effects of physiologically relevant amounts of arachidonic acid on membrane bilayers, alterations in membrane dynamics induced by arachidonic acid were investigated utilizing fluorescence and nuclear magnetic resonance spectroscopy. We demonstrate that decreases in the fluorescence steady-state anisotropy of diphenylhexatriene are induced by incorporation of physiologically relevant amounts (i.e., 5 mol%) of arachidonic acid into either phosphatidylcholine or plasmenylcholine membrane bilayers. Furthermore, examination of the motional dynamics of the bis-allylic protons in arachidonic acid by analyses of their spin-spin relaxation times demonstrated that these protons are more restrained when arachidonic acid is present as a substitutional impurity in plasmenylcholine vesicles than in phosphatidylcholine vesicles. Collectively, these results demonstrate that arachidonic acid, when present in physiologically relevant mole fractions, can modify the molecular dynamics of biological membranes and that the motional dynamics of arachidonic acid in membrane bilayers is influenced by the type of covalent linkage present in the proximal portion of the sn-1 aliphatic chain in host bilayer matrices.