To determine principal sources of eicosanoid production in glial cells, we analyzed the metabolites of arachidonic acid in cultured rat glial cells by use of reversed-phase, high-performance liquid chromatography and an on-line radioisotope detector. Prostaglandin D2, leukotriene B4, leukotriene C4, and 5-hydroxyeicosatetraenoic acid were present in cultures in which microglia appeared on a monolayer astrocytes. None were detected in culture dishes that contained only astrocytes, although astrocytes have been believed to be a main source of eicosanoid production in brain.