Phospholipid metabolism was studied in N1E-115 neuroblastoma and C6 glioma cells exposed to thapsigargin, a selective inhibitor of endoplasmic reticulum Ca(2+)-ATPase that raises the cytosolic free Ca2+ concentration [Ca2+]i. Thapsigargin caused only a transient increase of [Ca2+]i (< 1 min) in N1E-115 cells similar in magnitude and duration to agonist-induced calcium release mediated by inositol trisphosphate. Sustained elevation of [Ca2+]i due to influx of extracellular calcium, as occurs in most other cell lines including C6 cells, did not occur in N1E-115 cells. Increased uptake of inorganic phosphate (Pi) associated calcium influx was observed in C6 but not in N1E-115 cells. Thapsigargin affected phospholipid synthesis in both cell lines, most likely by inhibiting phosphatidic acid phosphohydrolase as indicated by diversion of [3H]oleic acid incorporation from triacylglycerol to phospholipid synthesis and stimulation of [32P]Pi incorporation into anionic phospholipids at the expense of phosphatidylcholine synthesis. The response to increased phosphatidate/phosphatidyl-CMP availability was cell specific. Thapsigargin (> 100 nM) selectively stimulated phosphatidylglycerol synthesis 20-30-fold in N1E-115 neuroblastoma cells while phosphatidylinositol synthesis was increased < 2-fold. In contrast, phosphatidylglycerol was not affected in C6 glioma cells and phosphatidylinositol synthesis was stimulated 8-fold by thapsigargin (> 1 microM). Agonist-stimulated calcium release did not increase phosphatidylglycerol synthesis in N1E-115 cells. Thapsigargin-stimulated phosphatidylglycerol synthesis and agonist-stimulated phosphatidylinositol synthesis could occur at the same time. Similar results were obtained with TMB-8, an inhibitor of intracellular Ca2+ release that decreases diacylglycerol utilization by blocking choline uptake and phosphatidylcholine synthesis without affecting resting [Ca2+]i. Thus [Ca2+]i does not directly mediate the effects of thapsigargin, TMB-8 or agonist stimulation on anionic phospholipid metabolism. These additional effects may limit the use of thapsigargin to assess Ca(2+)-dependence of phospholipid metabolism associated with Ca(2+)-mediated signal transduction.