The metabolites of arachidonic acid known as the leukotrienes are a class of lipid mediators which have potent and diverse biological effects in pulmonary tissue. Leukotrienes C, D, and E (LTC 4, LTD 4, and LTE 4) are known to be principal mediators of immunoglobulin E (IgE)-mediated hypersensitivity reactions in lung tissue. It is therefore important to develop reliable and quantitative isolation techniques for estimating levels of these mediators in tissue. In this study, LTC 4, LTD 4, and LTE 4 were separated from other arachidonate metabolites by organic extraction procedures. 5-Hydroxyeicosatetraeonic acid and leukotriene B 4 extract efficiently into the organic layer of aqueous:ether or aqueous:chloroform extractions, whereas arachidonate metabolites containing conjugated peptides (e.g., LTC 4, LTD 4, and LTE 4) failed to extract into these organic solvents. An extraction step was therefore developed that affords quantitative extraction of LTC 4, LTD 4, and LTE 4 into the organic phase of an isopropanol:ether:H 2O mixture. This step is the key for a two-step extraction method that isolates histamine, LTC 4, LTD 4, and LTE 4 with a recovery of 100, 85, 75, and 57%, respectively. One advantage of this separation procedure for obtaining these mediators by organic extraction is an ability to expediently process many samples. Furthermore, the leukotriene content of extracted samples can be analyzed using the guinea pig ileum bioassay without interference from vasoamines or platelet-activating factor. These later substances are eliminated from leukotriene-enriched fractions by this extraction process. When histamine and LTC 4 were added to supernatant fluids recovered from isolated lung tissue, they were quantitatively recovered using this extraction method. Histamine and LTC 4 were also incubated with control lung tissue and, after 10 min of incubation, 50% of the histamine and 40% of the leukotriene were recovered. There was extensive catabolism of the LTC 4 to LTD 4 and LTE 4. These results suggest that lung tissue contains potent catabolic pathways which rapidly metabolize the leukotrienes. Supernatant fluid from sensitized guinea pig lung tissue provoked by antigen to elicit an IgE-mediated hypersensitive reaction contained only 6.5 nmol histamine/g wet wt of tissue. Bathing fluid of the antigen-challenged tissue also contained LTD 4, a mediator not present in supernatant fluid from control tissue. The quantity of LTD 4 recovered from antigen-challenged tissue was less than 5 pmol/g wet of tissue. Levels reported here for extracted histamine and leukotriene were significantly lower than those previously reported using bioassays to analyze nonextracted samples.