The synthesis of cyclooxygenase products by cultured adrenergic neuronal (pheochromocytoma-12) cells was investigated by measuring both the extent of conversion of [3H]arachidonic acid to prostanoids and the immunoreactive prostanoid concentrations in the bathing buffer. Statistically significant amounts of arachidonic acid metabolites migrated with prostaglandins (PGs) E (81 ± 14 fmol) and F (68 ± 13 fmol) and thromboxane B (49 ± 12 fmol) on thin-layer chromatography plates after incubation of differentiated cells with 1 pmol of [3H]arachidonic acid. The conversion of arachidonic acid to these products was lower in undifferentiated cells, althougn PGE- and PGF-like metabolites were produced in significant amounts. Both immunoreactive PGE and thromboxane B were detected in the media of differentiated cells and their concentrations were elevated when the cells were exposed to arachidonic acid. The potential significance of the thromboxane production by pheochromocytoma-12 cells was investigated by examining the effect of a stable thromboxane mimetic, U46619, on potassium-stimulated norepinephrine release. The U46619 significantly enhanced norepinephrine release from potassium-depolarized cells. These results are indicative of PGE, PGF and thromboxane production by isolated adrenergic neuronal tissue. A thromboxane receptor agonist also was observed to potentiate norepinephrine release.