A method for the maximum recovery of prostaglandins from brain tissue with simultaneous recovery of neutral lipids and phospholipids was developed. Hexane:2-propanol was used to extract lipids from bovine brain. This method, which does not require a washing step to remove nonlipid contaminants, was compared to extraction according to Folch et al. [(1957) J. Biol. Chem. 226, 497-509] for efficiency of lipid extraction. Recoveries of prostaglandins were 12-37% greater with hexane:2-propanol than with the Folch extraction procedure with washing. The ratios of cholesterol to lipid phosphorus and absolute phospholipid recoveries were comparable for the two methods. A new elution sequence was devised for separation of lipid classes on silicic acid columns. The elution sequence was chloroform (neutral lipids and free fatty acids), methyl formate (prostaglandins and cerebrosides), acetone (remaining glycolipids), and methanol (phospholipids). Reverse-phase HPLC of the methyl formate fraction was used to separate the prostaglandins. The method permits simultaneous quantitative recovery of prostaglandins and phospholipids (which contain the 20:4(n-6) precursor for prostaglandin synthesis), and therefore allows changes in phospholipid composition and prostaglandin synthesis to be studied in the same tissue sample.