In cultured rat pituitary cells (GH4C1 cells) the ability of thyrotropin-releasing hormone (TRH) to stimulate phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) by a phospholipase C-type reaction was confirmed. The dose-response relationship for the TRH-stimulated phospholipase C was elucidated as was the relationship between the various inositol phosphates formed during the first few seconds after stimulation. The TRH-stimulated phospholipase C was subject to desensitization by repeated TRH treatment of cell cultures. This desensitization was dependent on the dose of TRH during preincubation. Following desensitization no decline in the levels of PIP2 was detected, even in the presence of decreased levels of PIP2 precursors. The TRH-stimulated phospholipase C activity was not attenuated following pretreatment with 12-O-tetradecanoylphorbol 3-acetate (TPA) to stimulate protein kinase C activity, and TRH also induced desensitization in the presence of the protein kinase C inhibitor polymyxin B. Thus, regulation of protein kinase C activity seemed not to be involved in the desensitization process. It is suggested that the ability of TRH to desensitize its own receptors and their link to phospholipase C, is mediated by the rise in intracellular calcium that is initiated by the TRH-receptor interaction.