We showed previously that polyenoic fatty acids with double bonds at carbon 5,8,11 are good substrates for the 5-lipoxygenase and also can be converted to LTC and dihydroxy acids. In order to determine whether all three double bonds are necessary for the 5-lipoxygenase-leukotriene pathway we studied 5,8,14-eicosatrienoic and 5,11,14-eicostrienoic acid. C14-labeled fatty acids were incubated with 10,000 × g supernatant of homogenate of rat basophilic leukemia (RBL-1) cells in the presence of Ca++ at 37°C. 5,11,14-Eicosatrienoic acid was not converted by the 5-lipoxygenase pathway and 5,8,14-eicosatrienoic acid was mainly converted to 5-hydroxy-6,8,14-eicosatrienoic acid (5-HETE). This monohydroxy was identified by UV spectrometry (UVmax 235 nm) and GC-mass spectrometry. Incubations with whole homogenate analyzed by HPLC and bioassay showed that no detectable LTC, LTD or LTE was formed. These data indicate that fatty acids which have double bonds at carbon 5 and carbon 8 are readily converted to the 5-hydroperoxide. However double bonds at carbon 5,8 and 11 are necessary for LTA biosynthesis. This study therefore extends the characterization of the double bond requirement of the 5-lipoxygenase-leukotriene pathway. The number of double bonds necessary at each step varies and increases with each step in the pathway.