We have developed a procedure for the microanalysis of the cholesterol and phospholipid concentrations and the fatty acid (including medium-chain fatty acids) composition of total lipid and phospholipid. One-milligram lipid samples were extracted from jejunal brush border membranes isolated from rats fed a diet containing 14% medium-chain triglycerides. The samples were analyzed by gas chromatography using an autosampler. We were able to identify 17 fatty acids in the total lipid and in the phospholipid fractions. The cholesterol and phospholipid contents of the membranes were 270 +/- 21 and 298 +/- 35 nmol.mg protein-1, respectively; thus the cholesterol/phospholipid ratio was 0.92 +/- 0.05, a value typically reported for the rat. Medium-chain fatty acids were not detected in the brush border membranes. Therefore, to demonstrate that medium-chain fatty acids were not lost during analysis, we analyzed the rat diet, and brush border membrane samples to which medium-chain triglycerides had been added. Recovery of medium-chain fatty acids from brush border membrane samples was 95.7 +/- 2.6%. The percentage of medium-chain fatty acids detected in the diet was not significantly different from the percentage when the diet was prepared. In summary, our procedure enabled a complete lipid analysis of very small biologic specimens.