Arachidonic acid can be converted to a large number of metabolites by various lipoxygenases, cyclooxygenase, and other enzymes. Because of the complex profiles of products formed by many types of cells, high-pressure liquid chromatography (HPLC) has proved to be an invaluable technique for their purification and analysis. In the present study we have developed improved methods for the analysis of complex mixtures of eicosanoids by HPLC using binary gradients containing trifluoroacetic acid (TFA), which allows considerable manipulation of the retention times of the cysteine-containing leukotrienes (LTs) LTC4, LTD4, and LTE4 relative to those of other eicosanoids. With a gradient between 0.003 and 0.005% TFA and a 4.6-mm-i.d. column of Spherisorb ODS-2, cysteinyl-LTs are very well resolved from one another and are separated as a group with retention times longer than those of all other major eicosanoids. These conditions can be used for the analysis of prostaglandin B2 (PGB2), LTB4, monohydroxyeicosatetraenoic acids (HETEs), and cysteinyl-LTs in only 30 min. Slightly longer analysis times must be used for the separation of more polar eicosanoids such as hydroxy metabolites of LTB4 and lipoxins. We have also developed methods for the analysis of eicosanoids using a midbore (3.2 mm i.d.) column containing Spherisorb ODS-2, which improves sensitivity and reduces solvent consumption. In this case higher concentrations (0.04 to 0.05%) of TFA have been used, resulting in retention times for cysteinyl-LTs between those of the cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid and the HETEs. This approach permits analysis of PGB2, LTB4, HETEs, and cysteinyl-LTs in only 20 min. Samples which also contain hydroxy-LTB4 and lipoxins can be analyzed in 40 min. The above techniques are highly reproducible and give baselines which are free of interfering peaks.