Cells of a mouse macrophage-like tumor cell line, J774.2, were incubated with 0.6μM radiolabeled mono- and di-hydroxyfatty acids. Monohydroxyfatty acid products of the neutrophil and platelet lipoxygenase pathways (5-HETE, 15-HETE, and 12-HETE) were rapidly taken up (42-64% of the counts cell associated at 1 min) and esterified into triglycerides and phospholipids. 5-HETE and 12-HETE were found in triglycerides and distributed among phospholipid classes while 50% of added 15-HETE was esterified into phosphatidyl inositol. Treatment of phospholipids from cells incubated with 5-HETE, 12-HETE, and 15-HETE with phospholipase A2 resulted in release of the respective monohydroxyfatty acid. HHT, a monohydroxyfatty acid product of the cyclooxygenase pathway, was taken up and esterified more slowly than the lipoxygenase products. In addition, HHT was not released when the phospholipids from cells incubated with HHT were treated with phospholipase A2. LTB4, a dihydroxyfatty acid product of neutrophil lipoxyegnase, was not taken up by J774.2 cells. The unique patterns of uptake and intracellular distribution of the different monohydroxyfatty acids suggests that the enzymes involved in the esterification of these compounds have substrate specificity and may also relate to the specific biologic effects of the compounds.