PAF (platelet-activating factor) is an endogenous mediator of endotoxin (LPS) shock and intestinal injury. In the present study we used an improved method to quantitate intestinal PAF after LPS injection. Both column and thin layer chromatography (TLC) were used to purify PAF. We found that using C18 column eluted sequentially with 10% acetic acid, ethyl acetate and 70% ethanol, yielded consistent results. TLC yielded falsely high PAF values, possibly from an unknown tissue lipid which co-migrated with PAF, or from toxic ingredients in the silica gel. Moreover, addition of optimal amounts of Tween-20 or ethanol in the bioassay samples enhanced PAF solubility and markedly improved PAF recovery. Lastly, dilution and heparinization of platelet-rich plasma greatly improved the sensitivity of the bioassay. The overall PAF recovery under these optimal conditions was 70-80%. We found that LPS (2-10 mg/kg, iv, 90 min) stimulated PAF production in the rat ileum, but not in the jejunum and colon. The difference in PAF production did not correlate to the numbers of sequestered neutrophils (reflected by myeloperoxidase levels) after LPS injection. This selective PAF production may account for the special vulnerability of the ileum to develop injury during endotoxemia.