Leukotrienes are lipid mediators that evoke primarily proinflammatory responses by activating receptors present on virtually all cells. The production of leukotrienes is tightly regulated, and expression of 5-lipoxygenase, the enzyme required for the first step in leukotriene synthesis, is generally restricted to leukocytes. Arachidonic acid released from the cell membrane of activated leukocytes is rapidly converted to LTA₄ by 5-lipoxygenase. LTA₄ is further metabolized to either LTC₄ or LTB₄ by the enzyme LTC₄ synthase or LTA₄ hydrolase, respectively. Unlike 5-lipoxygenase, these enzymes are expressed in most tissues. This observation previously has led to the suggestion that LTA₄ produced by leukocytes may, in some cases, be delivered to other cell types before being converted into LTC₄ or LTB₄. While in vitro studies indicate that this process, termed transcellular biosynthesis, can lead to the production of leukotrienes, it has not been possible to determine the significance of this pathway in vivo. Using a series of bone marrow chimeras generated from 5-lipoxygenase– and LTA₄ hydrolase–deficient mice, we show here that transcellular biosynthesis contributes to the production of leukotrienes in vivo and that leukotrienes produced by this pathway are sufficient to contribute significantly to the physiological changes that characterize an ongoing inflammatory response.