The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC₄-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10⁻⁵ M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)⁻¹ after 60 min incubation. In contrast, considerable thromboxane (TX) B₂ synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB₂ (mg protein)⁻¹). The synthesis of cysteinyl-LT was abolished by 5 × 10⁻⁵ M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10⁻⁶ M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC₄ and LTD₄ accounted for practically all the LTC₄-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE₄ could be detected. The results show that astrocytes may contribute to brain LTC₄ and LTD₄ synthesis. However, the cellular site of cerebral LTE₄ formation seems to be other than the astroglia.