Mammalian synaptic membranes appear to contain high proportions of specific, sn-1-stearoyl-2-docosahexaenoyl- and sn-1-stearoyl-2-arachidonoyl phosphoglycerides, but the structural significance of this is unclear. Here we used a standardized approach to compare the properties of homogeneous monolayers of the corresponding phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, and phosphatidic acids with those of control monolayers of sn-1-stearoyl-2-oleoyl- and sn-1-palmitoyl-2-oleoyl phosphoglycerides. Major findings were: 1), that the presence of an sn-2-docosahexaenoyl group or an sn-2-arachidonoyl group increases the molecular areas of phosphoglycerides by 3.8 A(2) (7%) relative to the presence of an sn-2-oleoyl group; 2), that the phosphorylcholine headgroup independently increases molecular areas by a larger amount, 7.1 A(2) (13%); and 3), that the dipole moments of species having an arachidonoyl moiety or an oleoyl moiety are 83 mD (19%) higher than those of comparable docosahexaenoic acid-containing phosphoglycerides. These and other results provide new information about the molecular packing properties of polyenoic phosphoglycerides and raise important questions about the role of these phosphoglycerides in synapses.