The human T lymphoblastoid cell line designated CCRF-CEM responds to phytohemagglutinin with a 3.7-fold enhancement of the 32PQ 4 incorporation into phosphatidylinositol. In myo-[2- 3H]inositol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a 3.3-fold accumulation of myo-[2- 3H]inositol phosphate during 15 min incubation at 37°C in the presence of 5 mM LiCl. Since Li + is a potent inhibitor of myo-inositol-1-phosphatase, the results indicate that phytohemagglutinin induces the hydrolysis of inositol lipids in CCRF-CEM cells. In 32 PO 4-prelabeled CCRF-CEM cells, phytohemagglutinin induced a breakdown of 28% of [ 32P]phosphatidylinositol 4,5-bisphosphate 40–60 s after the stimulation. The decrease of [ 32 P]phosphatidylinositol 4,5-bisphosphate was found as early as 10 s after the stimulation. This decrease was followed by an increased 32P-labeling of phosphatidic acid. In [2- 3H]glycerol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [ 3H] phosphatidic acid and [ 3H]diacylglycerol. The amount of [ 3H]phosphatidic acid in the stimulated cells was 3.7-times the control value at 2 min after the stimulation, whereas the amount of [ 3H]diacylglycerol in the stimulated cells was 1.5-times the control value at 5 min after the stimulation. In [ 3H 8]arachidonate-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [ 3H] phosphatidic acid; the amount was 2.5-times the control value at 2 min after the stimulation. Quinacrine (1 mM) caused 41% reduction in the amount of [ 3H] phosphatidic acid accumulated by the stimulation in [2- 3H]glycerol-prelabeled cells. Stimulation in a Ca 2+-free saline containing 1 mM EGTA caused 53% reduction in the amount of [ 3H]phosphatidic acid accumulated by the stimulation. The results presented in this paper indicate that a human T lymphoblastoid cell line, CCRF-CEM, responds to phytohemagglutinin with a rapid turnover of inositol lipids.