The relative proportions and compositions of the diacyl molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and diacylglycerol (DG) from rat retinal membranes were determined. Two membrane fractions were derived by discontinuous sucrose gradient floatation: rod outer segment (ROS) and 'rest of the retina' (ROR). ROR is defined as those membranes pelleted as 100,000 g following removal of the ROS. Diacylglycerols were prepared from PC, PE and PS by phospholipase C treatment and were converted into the corresponding 1,2-diacylglycerobenzoates (DGBZ). PI, PA, and DG were converted into 1,2-diacylglyceroacetates (DGAC) by acetolysis. The molecular species of the DGBZ and DGAC were resolved by reverse-phase HPLC and detected by UV absorption at 230 and 210 nm, respectively. Fatty acid methyl esters of PC, PE, PS, PI and DG from ROS and ROR were prepared and analysed by GLC. The fatty acid and molecular species patterns of PC, PE and PS were similar in both membrane fractions, although the levels of docosahexaenoic acid (22:6 omega 3) and 22:6-containing molecular species were lower in ROR than in ROS, PE and PS were enriched in 22:6 omega 3 and 18:0, an evidenced by the high levels of 18:0-22:6 and 22:6-22:6 molecular species. PC contained relatively more saturated and monoene species, such as 16:0-16:0, 16:0-18:0, 16:0-18:0, 16:0-18:1 and 18:0-18:1. The fatty acids and molecular species patterns of DG, PI and PA in ROS and ROR differed from those of PC, PE and PS.(ABSTRACT TRUNCATED AT 250 WORDS)