Primary cultures of neonatal rat brain polygonal astroglia, or cells of the C62B glioma line, were incubated with [1-14C]arachidonic acid (AA) in culture for 18 hr. In both culture systems, more than 80% of the added [1-14C]AA was taken up into cellular glycerolipids; less than 1% of the radioactivity in the cells was present in an unesterified form. When prelabeled C62B cells were stimulated with acetylcholine (ACh), there was a rapid accumulation of arachidonyl-phosphatidic acid (PA) accompanied by a liberation of [1-14C]AA. A variety of other neurotransmitters failed to activate this response in C62B glioma cells. In contrast to the agonist specificity of the response in C62B glioma cells, primary astroglia generated PA and liberated [1-14C]AA in response to several neurotransmitters (i.e., ACh, norepinephrine, glutamate, and histamine) Treatment of astroglia with a combination of norepinephrine, ACh, and histamine resulted in a greater production of PA and free [1-14C]AA than did treatment with any one of these neurotransmitters alone. The results suggest that cultures of astroglia can respond to several different neurotransmitters with specific changes in AA and PA metabolism. Thus, a variety of neurotransmitters initiate cascades of lipid metabolism which may be of physiological significance in glial function.