We have shown that there is an inositol (Ins) lipid pool in cloned rat pituitary tumor (GH3) cells that is hydrolyzed in response to thyrotropin-releasing hormone (TRH) and an unresponsive pool. Because others have suggested that incorporation of [3H]Ins by base exchange may not occur uniformly into Ins lipids in other cell types, we established conditions using permeabilized cells under which labeling occurs by Ins-phosphatidylinositol (PI) exchange in the absence of de novo PI synthesis to further characterize these pools in GH3 cells. In permeabilized cells incubated in buffer containing 10 mM Mg2+ and 0.1 mM CMP, [3H]Ins incorporation into lipids occurred by base exchange only. This was so because: 1) [3H]Ins incorporation into lipids displayed properties similar to that for release of 3H-labeled Ins by unlabeled Ins from PI in cells prelabeled in situ prior to permeabilization; and 2) there was no change in PI mass under these conditions. In permeabilized cells incubated in buffer with 0.1 mM [3H]Ins for 60 min, incorporation was 0.61 +/- 0.05 nmol of [3H]Ins/10(6) permeabilized cells, which amounted to 35% of PI, while the level of PI, measured as nonradioactive phosphorus, was 94 +/- 8.0% of control. Permeabilized GH3 cells were responsive to TRH. In cells prelabeled in situ and then permeabilized, TRH stimulated an increase in 3H-labeled Ins phosphates (IPs) in 20 min which was 10% of 3H radioactivity initially present in lipids. This increase in 3H-labeled IPs was 6.3 times the 3H radioactivity present in phosphatidylinositol 4,5-bisphosphate prior to stimulation. When prelabeled cells were exchanged with unlabeled Ins after permeabilization there was only a 10-16% decrease in 3H-labeled IP accumulation stimulated by TRH even though 3H-labeled lipids decreased to 52% of control. TRH did not affect labeling by [3H]Ins-PI exchange. In cells labeled by base exchange after permeabilization TRH stimulated a very small increase in 3H-labeled IPs of only 0.21 +/- 0.02% of 3H-labeled lipids in 20 min or only 7% of the 3H radioactivity in phosphatidylinositol 4,5-bisphosphate. These data show that in permeabilized GH3 cells base exchange can occur in the absence of de novo PI synthesis and that lipids that are preferentially labeled by base exchange comprise a pool that is less responsive to TRH than total Ins lipids.