Quantities of 1-palmitoyl 2-docosahexaenoyl phosphatidylcholine (16:0/22:6-PC or PDPC) increase from 24 to 40 weight percent as a consequence of cold acclimation in mitochondrial membranes of rainbow trout liver (J. Comp. Physiol. 156, 665-674, 1986). The present study was undertaken to assess the impact of such a large change in the proportions of a single molecular species on the fluidity, lateral packing (as sensed by phospholipase A2), and permeability of biological membranes. These properties were examined in multilamellar liposomes prepared from binary mixtures of dipalmitoyl phosphatidylcholine (DPPC) and PDPC in proportions increasing from 10 to 40 mole% PDPC. Glucose permeability was positively correlated with both assay temperature and PDPC content. The temperature dependence of Na+ permeability declined steadily as the mole fraction of PDPC increased; consequently, sodium permeability was positively correlated with PDPC content at 5 degrees C, but inversely correlated at 20 degrees C. Phospholipase A2 activity was independent of both assay temperature and vesicle composition. Vesicles of all compositions displayed a single transition in the temperature dependence of 1,6 diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization, which shifted to lower temperature and broadened as proportions of PDPC increased. At temperatures below the transition, fluidity was positively correlated with the mole fraction of PDPC, but interfacial and deeper regions of the bilayer were affected differently by variations in PDPC content. Nonelectrolyte permeability was the only index of membrane structure or function to be significantly correlated with the fluidity of the bilayer interior. The tendencies of PDPC to both fluidize the membrane and to reduce the temperature sensitivity of electrolyte permeation may promote the adaptation of membrane function to low temperature.