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The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.