We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n - 9) and stearate/eicosatrienoate (n - 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n - 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.