We report that there are distinct thyrotropin-releasing hormone (TRH)-responsive and -unresponsive pools of inositol (Ins) lipids in rat pituitary tumour (GH3) cells, and present evidence that the size of the responsive pool is determined by the number of activated TRH-receptor complexes. By use of an experimental protocol in which cycling of [3H]Ins is inhibited and resynthesis occurs with unlabelled Ins only, we were able to measure specifically the effects of TRH on the hydrolysis of the Ins lipids present before stimulation. A maximally effective dose of TRH (1 microM) caused a time-dependent decrease in 3H-labelled Ins lipids that attained a steady-state value of 42 +/- 1% of the initial level between 1.5 and 2 h. After 2 h, even though there was no further decrease in 3H-labelled Ins lipids, and no increase in [3H]Ins or [3H]Ins phosphates, turnover of Ins lipids, as assessed as incorporation of [32P]Pi into PtdIns, continued at a rate similar to that in cells incubated without LiCl or unlabelled Ins. These data indicate that Ins lipid turnover was not desensitized during prolonged TRH stimulation. Depletion of lipid 3H radioactivity by TRH occurred at higher TRH doses on addition of the competitive antagonist chlordiazepoxide. Addition of 1 microM-TRH after 3 h of stimulation by a sub-maximal (0.3 nM) TRH dose caused a further decrease in 3H radioactivity to the minimum level (40% of initial value). We propose that the TRH-responsive pool of Ins lipids in GH3 cells is composed of the complement of Ins lipids that are within functional proximity of activated TRH-receptor complexes.